ŒARACTERIZATION OF RECOMBINANT VACCINIA VIRUSES EXPRESSING THE LASSA VIRUS GLYCOPROTEIN GENES (11 Lc

Thelma LescottMaster of Philosophy 1994

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SUMMARYLassa virus (LV) is a member of the Arenaviridae

family and categorized as a hazard group 4 virus by the Advisory Committee on Dangerous Pathogens. These viruses require maximum containment facilities for infectious virus studies. Consequently the virus and the disease have been very poorly researched.

This study describes the construction and characterization of recombinant vaccinia viruses expressing LV glycoproteins prepared with two strains of vaccinia . virus; Lister and Western Reserve. The LV genome encodes two glycoprotein genes, G1 and G2. Both glycoproteins have been expressed, identified and characterized as being authentic viral proteins using anti-LV human antisera and mouse monoclonal antibodies.

To assess if the recombinant vaccinia viruses would protect animals from a lethal challenge of LV, guinea pigs were immunized with these recombinant vaccinia viruses, then challenged 56 days after vaccination with a lethal dose of LV. A recombinant vaccinia virus expressing the LV nucleoprotein, which had been shown to protect in previous studies, was included for comparison. Various parameters of LV infection were monitored for 35 days post challenge. Although the recombinant vaccinia viruses protected guinea pigs from the lethal LV challenge, examination of disease profiles in vaccinated and unvaccinated animals revealed that protected animals still had clinical evidence of LV disease,

In the guinea pigs immunized with the recombinant vaccinia viruses, the effects of the disease were moderated in most animals surviving LV challenge. Weight loss, fever and haematological factors were mild to moderate in vaccinated animals. A correlation has been established in human patients with Lassa Fever between increased serum aspartate aminotransferase (AST) levels and a greater risk of death. The AST levels in these vaccinated guinea pigs remained at moderate levels, whereas the levels in the control guinea pigs were greatly increased.

11

LIST OF CONTENTS

TitleSummaryList of Contents Acknowledgements List of Tables List of Figures Abbreviations

Pagenumber

1iiiviiiixXxii

CHAPTER

LITERATURE REVIEW

SECTION 1: THE ARENAVIRUSES

INTRODUCTION TO THE ARENAVIRIDAE1. Classification

B. BIOLOGY OF THE ARENAVIRUSES1. Diagnosis2. Pathogenesis3. Hosts4. Lymphocytic choriomeningitis5. Lassa fever6. Junin7. Machupo8. Antigenic relationships

C. BIOCHEMISTRY AND MOLECULAR BIOLOGY1. Virion structure and virus replication2. Genome3. Major viral structural proteins

D. LASSA VIRUS1. Animal model2. Antiviral therapy3. Vaccines4. Studies using vaccinia virus and

LV gene recombinants

2469

101314 16

202326

31353637

111

SECTION 2: RECOMBINANT VACCINIA VIRUSES

A. ERADICATION OF SMALLPOX1. Classification 442. History 453. Pathogenesis 474. W.H.O. eradication programme 495. Variola virus eradication 53

B. VIRUS REPLICATION1. Morphology and virion structure 542. Virus entry into cells 563. Gene expression 574. DNA replication 605. Virion assembly 61

C. GENERATION OF RECOMBINANT VACCINIA VIRUSES1. Procedure 632. Recombinant selection 653. Factors affecting expression 674. Uses of recombinant vaccinia viruses 69

D. VACCINIA AS A VECTOR1. History 702. Attenuation of vaccinia virus 73

E. AVIAN POXVIRUSES AS VECTORS 76

F. HOST IMMUNE RESPONSE TO VACCINATION 77

SECTION 3: AIMS OF THESIS 81

IV

CHAPTER 2

MATERIALS AND METHODS

A. CELL CULTURE, VIRUSES AND ANTISERA1. Cells 822. Viruses 823. Antisera and monoclonal antibodies 83

B. INFECTIVITY TITRATIONS 83

C. TRANSFECTION AND SELECTION OF RECOMBINANT VIRUSES1. Transfection of cells 842 . Selection of recombinants 84

D . INDIRECT IMMUNOFLUORESCENCE1. Permeabilized cells 852. Non-permeabilized cells 86

E. RADIOLABELLING AND POLYACRYLAMIDE GELELECTROPHORESIS 86

F. PURIFICATION OF VIRUS FOR INOCULATION OF ANIMALS 87

G. A.C.D.P. CATEGORY 4 LABORATORIES1. Virus laboratory 882. ACDP level 4 animal facilities 90

H. ANIMAL STUDIES1. Guinea pigs 912. Vaccination and challenge 913. Haematology and biochemistry 924. ELISA 935. Virus studies 94

V

CHAPTER 3RESULTS

SECTION 1: IN VITRO STUDIES

A. INTRODUCTION 95

B . RESULTS1. Expression of viral glycoproteins,

inside infected cells 972. Expression of viral glycoproteins,

on the surface of infected cells 1003. Radiolabelling of viral glycoproteins 1004. Reactivity of monoclonal antibodies

to Lassa virus glycoproteins with Arenavirus strains 103

5. Growth of recombinant viruses indifferent cell lines 106

C. DISCUSSION 108

SECTION 2 ; ANIMAL STUDIES

A. INTRODUCTION1. Experimental design 113

B . RESULTS1. Post-immunisation status of animals 1142. Antibody levels to vaccinia virus

and LV 1153. Post-challenge status of animals 1154. Animals surviving LV challenge 1175. Weight 1176. Temperature 1217. Haematology 1238. Biochemistry 1319. Post challenge anti-LV antibody levels 133

10. Virus levels 135C. DISCUSSION 13 8

VI

CHAPTER 4

CONCLUSION 142

REFERENCES 147

Vll

ACKNOWLEDGEMENTS

I would like to thank my supervisors Dr. Alan Barrett and Dr. David Kelly for their help, encouragement and guidance throughout this project.I am especially grateful to Dr. Graham Lloyd and Dr. Chris Clegg at CAMR. Chris for supplying the plasmid construct. Graham for sharing his knowledge and expertise.My thanks also go to the support staff at CAMR for caring for the animals, Tracy Benford for cell culture maintenance and Steve Eley for word processing advice.M.o.D. are gratefully acknowledged for financial support.My children, Richard and Paula, are thanked especially for their support and co-operation.

V l l l

LIST OF TABLES

Chapter 1.Pagenumber

Table 1. Members of the Arenaviridae.Table 2. Clinical symptoms of human

arenavirus infections.Table 3. The natural hosts of individual

arenaviruses.Table 4. Comparison of potential N-linked

glycosylation sites in arenavirus glycoproteins.

Table 5. Protection of guinea pigs against a challenge of lO^pfu LV with recombinant vaccinia viruses.

Table 6. Factors affecting the eradication of smallpox.

Table 7. Advantages and disadvantages of vaccinia virus as a potential vaccine vector.

33

41

52

72

Chapter 3. Table 8.

Table 9.

Table 10.

Table 11.

Table 12.

Table 13. Table 14.

Reaction of arenaviruses with monoclonal antibodies (MAbs) specific for LV glycoproteins, G1 and G2.Infectivity titres of vaccinia and recombinant vaccinia viruses in CVl and RK13 cell lines.Infectivity titres of vaccinia and recombinant vaccinia viruses after one passage in RK13 cells.Infectivity titres of vaccinia viruses after passage in either CVl or RK13 cells.Serum IgG antibody levels to vaccinia virus.Animals surviving LV challenge.Data summary of immunized guinea pigs following challenge with Lassa virus.

105

107

109

110

116118

137IX

LIST OF FIGURESChapter 1.

Figure 1. Map of South America showinggeographic distribution of New World arenavirus isolates.(after Tesh et al., 1994)

Figure 2. Map of Africa showing geographicdistribution of Old World arenavirus isolates.(after Lloyd, 1983)

Figure 3. Schematic diagram of an arenavirus particle, (after Bishop, 1990)

Figure 4. Diagram representing translation of the arenavirus genome.(after Clegg, 1992)

Figure 5. Phylogenetic tree showing amino acid sequence relationships of arenavirus nucleoproteins.(after Clegg, 1992)

Figure 6. Dendrogram of relationships between arenavirus glycoprotein nucleotide sequences.(after Clegg et al., 1990)

Figure 7. Numbers of countries in which smallpox was endemic between 1920 and 1977. (after Fenner, 1986)

Figure 8. Vaccinia virus replication cycle.(after Moss, 1990a)

Figure 9. General procedure for producing vaccinia virus recombinants.

Pagenumber

17

19

21

25

27

29

50

58

64

Chapter 2

Figure 10. ACDP level 4 containment laboratory at CAMR, Porton Down. 89

X

Chapter 3.Figure 11. Indirect immunofluorescence of WRGPC

infected, acetone fixed cells. 98Figure 12. Indirect immunofluorescence of WR

infected, acetone fixed cells. 98Figure 13. Indirect immunofluorescence of LISGPC

infected, acetone fixed cells. 99Figure 14. Indirect immunofluorescence of LIS

infected, acetone fixed cells. 99Figure 15. Indirect immunofluorescence of WRGPC

infected, non-fixed cells. 101Figure 16. Indirect immunofluorescence of WR

infected, non-fixed cells. 101Figure 17. Indirect immunofluorescence of LISGPC

infected, non-fixed cells. 102Figure 18. Indirect immunofluorescence of LIS

infected, non-fixed cells. 102Figure 19. LV glycoproteins labelled with [ S] -

methionine and immunoprecipitated withhuman anti-LV serum. 104

Figure 20. Body weight of vaccinated guinea pigsfollowing challenge with LV. 120

Figure 21. Rectal temperature of vaccinated animalschallenged with LV. 122

Figure 22. Platelet counts in vaccinated animalsafter challenge with LV. 124

Figure 23. Neutrophil counts in vaccinated guineapigs following challenge with LV. 126

Figure 24. Lymphocyte counts in guinea pigsfollowing challenge with LV. 128

Ficfure 25. Leukocyte counts in vaccinated guineapigs following challenge with LV. 13 0

Figure 26. Serum AST levels in vaccinated guineapigs following challenge with LV. 132

Figure 27. Serum ALT levels in vaccinated guineapigs following challenge with LV. 134

Figure 28. Serum anti-LV antibody titres in guineapigs after challenge with LV. 13 6

xi

ABBREVIATIONS

ACDPASTALTCAMRCDCELISAFITCG1G2GPCIgGlUkDaLCMLFLISLISGPC

LISN

LVMAbmoiNPBSPBSTPCpfuRNATCID50WHOWRWRGPC

Advisory Committee on Dangerous Pathogens Aspartate aminotransferase Alanine aminotransferaseCentre for Applied Microbiology Research Centers for Disease ControlEnzyme-linked immunosorbent assayFluorescein isothiocyanateLassa virus glycoprotein of approximate molecular weight 45 kDaLassa virus glycoprotein of approximatemolecular weight 3 8 kDaLassa virus precursor glycoproteinImmunoglobulin G International unitskilodaltonLymphocytic choriomeningitis Lassa feverLister strain of vaccinia virus Recombinant vaccinia virus prepared with Lassa virus precursor glycoprotein and the Lister strain of vaccinia virus Recombinant vaccinia virus with an insert of Lassa virus nucleoprotein in vaccinia virus (Lister strain)Lassa virusMonoclonal antibody multiplicity of infectionLassa virus nucleoproteinPhosphate buffered saline0.05% Tween 20 in PBS post challenge plaque forming unitsribonucleic acidtissue culture infectious dosesWorld Health Organisation Western reserve strain of vaccinia virus Recombinant vaccinia virus of Lassa virus precursor glycoprotein and Western reserve strain of vaccinia virus

X l l

CHAPTER 1

LITERATURE REVIEW

SECTION 1. THE ARENAVIRUSES

A. INTRODUCTION TO THE ARENAVIRUSES

1. ClassificationThe first arenavirus was identified in 1933 by-

Armstrong and Lillie (Armstrong and Lillie, 1934). It was given the name lymphocytic choriomeningitis (LCM) virus after causing disease in mice and monkeys, and was eventually identified as causing aseptic meningitis in man (Rivers and Scott, 1935). Subsequently, the Arenaviridae family was formed on the basis of morphology and biochemical properties. The family is named after the sandy (Latin arenosus) appearance of the virus particles (Rowe et al., 1970a). This "sandy" appearance is caused by the incorporation of host cell ribosomes into the viruses. These ribosomes give thin sections of virus particles a granular or sandy structure. Listed in Table 1 is LCM virus, the prototype, and 16 other viruses that currently comprise the family. Biochemically, the virion structure of all the members of the family comprises three virus structural proteins surrounding the ribonucleic acid (RNA) genome. A more detailed description will be given in the section on biochemistry and molecular biology later in this chapter (section 1. C.2) .

From Table 1 it can be seen that there are two geographically distinct groups, often known as the Old World (African isolates) and the New World (South and Latin American isolates) viruses. The latter is also known as the Tacaribe complex. By serological tests LCM virus is more closely related to the Old World viruses than those of the New World. Five of the viruses (Lassa, Mopeia, Mobala, Junin and Machupo) listed in Table 1 are classified as hazard category 4 agents by the Advisory Committee on Dangerous Pathogens (ACDP). Viruses in this hazard group cause severe human disease and are a serious hazard to laboratory workers. With category 4 viruses there is also a high risk of spread in the community and little or no effective treatment available. Although the immunology of LCM virus has been extensively studied (Oldstone, 1987), there is little information available on the category 4 arenaviruses.

B. BIOLOGY OF THE ARENAVIRUSES

1, DiagnosisImmunological diagnosis of acute arenavirus

infections was, in the past, achieved by complement fixation tests using acute and convalescent serum samples (Johnson, 1989). More recently immunofIncrescent antibody assays and enzyme-linked immunosorbent assays (Lewis et al., 1975; Niklasson et al., 1984) have been used as they have the advantage of early detection of viral antigens.

TABLE 1. MEMBERS OF THE ARENAVIRIDAE

OLD WORLD

Virus Reference and year isolated

Geographicdistribution

ACDP * category

Lymphocyticchoriomeningitis

Armstrong1934

World-wide 3

Lassa Buckley1970

WesternAfrica

4

Mopeia Wulff 1977 SouthernAfrica

4

Mobala Gonzalez1983

CentralAfrica

4

ippy Swanpoel1985

CentralAfrica

2

Sku Kiley 1986 CentralAfrica

2

NEW WORLD

Tacaribe Downs 1963 Trinidad 2Junin Parodi 1959 Argentina 4Machupo Johnson 1965 Bolivia 4Amapari Pinheiro 1966 Brazil 2Pichinde Trapido 1971 Colombia 2Parana Webb 1970 Paraguay 2Latino Casals 1975 Bolivia 2Flexal Pinheiro 1977 Brazil 2Tamiami Calisher 1970 Florida 2Guanarito Salas 1991 Venezuela 3Sabia Coimbra 1994 Brazil 3

ACDP is an abbreviation for the Advisory Committee on Dangerous Pathogens. This committee issues guidance on the categorisation and containment of pathogens in the U.K. There are 4 categories assessed on the degree of hazard. Four is the most hazardous; one is the least hazardous.

virus isolation was necessary before a specific diagnosis of the disease could be made, but is now often unnecessary. Arenaviruses grow to reasonable titres in several mammalian cell cultures with Vero cells being the most commonly used cell line for isolation and identification. Lassa virus (LV) has been recovered from throat washings and urine of patients (Monath et al., 1974a) and in fatal cases of Lassa fever (LF), liver has been used to confirm diagnosis from human post-mortem samples. The use of the polymerase chain reaction has also been successfully applied to detecting arenaviruses (Clegg and Lloyd, 1991).

2. PathogenesisAfter the initial onset of infection, all

arenaviruses progress to multiorgan infections with divergent pathology and pathogenesis. The diseases caused by LCM, Lassa, Junin and Machupo viruses will be discussed in detail later, but a summary comparing the clinical symptoms in humans is shown in Table 2 (McCormick, 1990). A demonstration of the diversity of the disease caused by the viruses in the group is given by comparing the mortality data. Argentinian Haemorrhagic Fever (AHF), caused by Junin virus, has case fatality rates of 5-16%, Bolivian Haemorrahagic Fever (BHF), caused by Machupo virus, of 5-25%, LF of 20%; whereas deaths are rarely reported from LCM infections. It can be seen in Table 2 that many of the symptoms due to

TABLE 2. CLINICAL SYMPTOMS OF HUMAN ARENAVIRUSINFECTIONS

CLINICAL SYMPTOMS VIRUSLCM Lassa Junin/

MachupoFever - ++ ++Headache + + ++ ++Myalgia + + ++ ++Lower back pain + + +Sore throat + ++ -

Pharyngeal exudate - +++ -

Retrosternal pain + ++ +Vomiting/diarrhea - ++ -

Erythema + + +++Cough + ++ -

Jaundice - - -

Bleeding - + ++Shock - +++ ++Acute respiratory disease - ++ -

Meningitis + + - -

Encephalopathy - + +Deafness - + +Thrombocytopenia - - ++Leukopenia ++ ++ ++

Fatality no yes yes

KEY: - no symptoms+++ } degree of symptoms ranging from mild(+)+++ to severe (+++).

different arenavirus infections are similar, especially with LF and the other haemorrhagic fever viruses. In LCM infections, the disease is usually much less severe, although LCM virus is the only arenavirus that causes meningitis. LF is the only disease which affects the respiratory tract. There is often more viraemia in LF patients for a longer period of time and permanent deafness has been documented as a result of Lassa virus (LV) infections (White, 1972).

3. HostsThe natural hosts and reservoirs of the arenaviruses

causing human disease are often rodents. In Table 3 is a list of arenaviruses and the species from which they are commonly isolated. Although there are over thirty rodent families, arenaviruses are associated with only two of the families, Muridae and Cricetidae. The genera Mus, Rattus, Mastomys and Praomvs are comprised of rats and mice in the family Muridae and are found only in the Old World. Similarly, the genera Akodon. Calomvs. Neacomvs. Orvzomvs. Sigmodpn, and Thomasomvs consist of mouse-like and rat-like rodents only found in the New World (Johnson, 1989). Many of these rodents appear very similar, but their habitats, habits and life histories differ considerably. There is also an exception to the arenavirus host range of only mouse-like and rat-like rodents. Tacaribe virus, from the New World complex, has been isolated from the fruit-eating bat Artibeus, which

TABLE 3 THE NATURAL HOSTS OF INDIVIDUAL ARENAVIRUSES

OLD WORLDVirus Natural host *

Lymphocyticchoriomeningitis

Mus mus cuius

Lassa Mastomys natalensis

Mopeia Mastomys natalensis

Mobala Praomys jackson

Ippy Arvicanthis speciesSku Mastomys species

NEW WORLDTacaribe Artiheus species (bat)Junin Calomys laucha

Calomys musculinus

Machupo Calomys callosus

Amapari Oryzomys goeldi Neacomys guinae

Pichinde Oryzomys alhigularis

Parana Oryzomys buccinatus

Latino Calomys speciesTamiami Sigmodon hispidus

Oryzomys palustris

Flexal Oryzomys speciesGuanarito Sigmodon alstoni

Sabia Unknown

* The predominant natural host is listed for each virus.

is the most common host of the virus. The natural host of the recently isolated Sabia virus has not yet been identified, however it is thought to be a rodent (Coimbra et al., 1994).

Frequently an arenavirus infection in the rodent host is persistent, which although not detrimental to the host, will result in infectious virus being shed into the environment via the urine, faeces and saliva. Airborne infectivity has also been demonstrated in laboratory animals by accidental transmission involving animal husbandry. LCM virus was transmitted from mice to monkeys when LCM infected mice were kept in the same room as healthy monkeys (Peters et al., 1987). Subsequently, monkeys died from LCM and minute quantities of airborne LCM virus were detected in the room air, but only when the bedding from mouse cages was handled. LV has also been shown to spread from one set of laboratory animals to another in similar circumstances. Aerosol studies on LV (Stephenson et al., 1984) and the haemorrhagic fever viruses (Samoilovich et al., 1984) have shown their aerosol stability and infectivity to be high. Thus, transmission of the virus by this route from rodent to human appears very plausible. Although the diseases of LCM, Lassa, Junin and Machupo will be outlined in more detail below, infections vary from mild to severe for each virus and factors such as virus strain, host genotype and the immunological status of the host are all

important parameters that contribute to the virus infection.

4. Lvmphocvtic choriomenincritisThree types of illness are manifested in humans by

LCM virus. These are aseptic meningitis, an influenza­like disease and a meningo-encephalomyelitic illness (Lehmann-Grube, 1971). When the virus was first identified it was assumed to be the only causative agent of acute aseptic meningitis or Wallgren disease (Rivers and Scott, 1935). Subsequently, this has proved to be incorrect and LCM virus is responsible for only a small proportion of these cases. One study, involving American military personnel (Meyer et al., 1960), showed that over an eighteen year period only 8% of 1,600 central nervous system (CNS) cases were identified as being caused by LCM virus.

The influenza-like and meningeal type illnesses are most frequently caused by LCM virus; both having an incubation period of 6-13 days. Symptoms of the meningeal type include headache, stiff neck and nausea, lasting for periods of 2 weeks or longer. The severity of the illness can, however, result in hospitalization and prolonged convalescence. With the influenza-like type of illness, symptoms of fever, malaise, muscular pains and bronchitis are common. Most specifically identified LCM cases follow a benign course and fatalities are few, usually only after the virus has invaded the CNS.

The geographical distribution of the virus is almost world-wide, even though there are doubts about its existence in Australia (Lehmann-Grube, 1971).Principally, the house mouse (Mus muscuius) is the main animal reservoir of the virus. There appears to be some seasonal variation in the disease in man, case numbers being greater in the winter months, when mice move indoors to escape the cold, thus enabling more frequent contact with man (Ackerman et al., 1964). Exactly how the virus is transmitted from mouse to man is unknown, but possible routes are from infected urine and via cuts and abrasions (Johnson, 1989).

Laboratory personnel have been infected with LCM virus when working with mice, hamsters and monkeys (Lewis et al., 1965; Sulkin and Pike, 1969). LCM virus was isolated from hamsters in commercial breeding colonies in West Germany (Forster and Wachendorfer, 1973) and studies have shown pet hamsters (Emmons et al., 1974) and white mice (Gregg, 1975) to be sources of human infections. The persistence of the virus in hamsters is less prolonged than in mice: virus in hamsters has only been detected for 2 to 3 months after infection, whereas virus is shed throughout the life-span of the mouse (Johnson,1989).

5. Lassa feverLF has an incubation period of seven to eighteen

days before the onset of fever, headache and malaise (Monath, 1975) . Fever ranges from 38-41°C and can last for

10

up to three weeks. Many patients also have muscular aches, abdominal pains, nausea, vomitting and diarrhoea. Seventy per cent of patients develop pharyngitis and thirty per cent develop conjunctivitis (McCormick, 1987) . Hepatitis and high serum levels of aspartate aminotransferase (AST) correlate with fatalities. Only 15-2 0% of cases result in bleeding, usually in the gums and nose, but gastrointestinal and vaginal bleeding occurs in severe cases.

Although the disease was first described in West Africa in 1956 (McCormick and Johnson, 1978), it was not until an outbreak in Nigeria in 1969 that the disease was associated with the virus. Since the cases in Nigeria, cases of LF have also been reported in Liberia and Sierra Leone. Serological surveys indicate that LV probably also occurs in Guinea, Central African Republic, Mali,Senegal, Cameroon and Benin. It is possible that the disease may be more widespread throughout Africa, but as it is difficult to diagnose and often confused or accompanied by malaria, this proposal has as yet to be proven.

There have been extensive studies to investigate the reservoir of LV in West Africa. In Sierra Leone in 1972 the tissues of 325 rodents and bats were tested for LV virus. Only ten of 46 Mastomvs natalensis samples contained virus (Monath et al., 1974a). Mastomvs natalensis is one of the most common rodents in Africa and both species of Mastomvs that occur in West Africa

11

are carriers of LV (McCormick, 1987) . In Southern Africa two different species of Mastomvs natalensis are common and these have been shown to be persistently infected with Mopeia virus, a virus closely related to LV (Johnson et al., 1981). The habitat of these rodents is usually close to villages and in houses, so that they live in close proximity with humans. Spread of the virus from rodent to man is probably from contaminated food and aerosol transmission. Infection rates are high in humans when the rodent population is large and where rodents are caught, cooked and eaten.

As well as rodent to human transmission, LV is also spread from person to person, although this route is less common (Keenlyside et al., 1983). The first identified cases of LF occurred in 1969, when a Lassa patient passed the virus to two nurses after admission to hospital and two of the three infected patients subsequently died. Transmission in hospital could have been by either direct contact, the airborne route or by clinical instruments. Direct contact with infected blood is a possible means of infection in hospital personnel, because virus concentrations have been shown to be high (>10‘pfu/ml) in blood samples from seriously ill LF patients (Johnson, 1989). Also there have been infections acquired through a cut at autopsy (White, 1972) and by a nurse with a cut finger (Frame et al., 1974). Laboratory accidents have also occurred in the United States (Leifer et al., 1970) .

12

Person to person spread of LV is also common in the household environment.

6. JuninThe disease caused by Junin virus was first

recognised in 1943 and because of its geographical location and the illness characteristics, subsequently became known as Argentinian Haemorrhagic Fever (AHF). There is an incubation period of seven to sixteen days (Maiztegui, 1975) with a gradual increase of malaise, fever, headache, retro-orbital and muscular pain. Anorexia, nausea and vomitting frequently occur, but unlike LF, the respiratory system and throat are usually unaffected. With LCM there are no haemorrhagic complications and only rarely with LF, however almost half the patients with AHF have problems with bleeding. Haemorrhaging often occurs from the gums and nose or into the skin forming a haemorrhagic rash (Mettler,1969). Neurological problems also occur in almost 50% of AHF patients and the case fatality rate varies between 3 and 15%, with almost no clinically inapparent infections.

AHF was first recognized in the northwest of Buenos Aires in the 1950's. Areas of infection have subsequently spread to the surrounding provinces of Cordoba and Santa Fe, with over 20,000 cases of the disease being reported from 1958 to 1984 (Maiztegui et al., 1986). The disease pattern is seasonal with increases in case numbers during late summer (February) and reaching a peak in autumn

13

(May). This coincides with maize harvesting and an increase in the rodent population. Although in human populations, both sexes are susceptible to the disease, almost 80% of AHF cases have been in adult males employed in agriculture.

There are two species of rodent that are the main reservoir for Junin virus and these are Calomys musculinus and Calomvs laucha (Maiztegui et al., 1986). Both share the habitat of grain fields and the principle route of transmission of the virus to man appears to be from contaminated grain, dust and through skin abrasions. Junin virus has also been isolated from the rodents Akodon azarae and occasionally Mus musculus (Sabattini et al., 1977). There have also been reports of virus isolation from mites, although it has not been established that mites are involved in transmitting the virus from rodents to man.

7. MachupoThe diseases caused by Junin virus and Machupo virus

are very similar (Johnson et al., 1967). As with AHF, the disease caused by Machupo virus obtained its name from Bolivia, the area of the first recognised epidemic, and subsequently became known as Bolivian Haemorrhagic fever (BHF). The incubation period is from seven to fourteen days, which is followed by a disease characterised by a high fever lasting for at least five days. Bleeding occurs in almost 3 0% of patients, although blood loss

14

itself is not life threatening. With 50% of cases,(

tremors of the tongue and hands occur, which can progress to extensive neurological disorders. There is frequently a long convalescent period manifesting itself in severe weakness and neurological malfunctions. A case fatality rate of between 5 and 30% has been recorded, depending on the outbreak.

BHF was first reported in Bolivia in 1959 and the virus first isolated in 1964 (Johnson et al., 1965). Originally the outbreaks of disease were in rural areas, but subsequent affected areas have been in towns and villages. In earlier disease patterns adult males with agricultural occupations were principally infected, then with later patterns there appeared to be no correlation between disease, sex, age and occupation (Mackenzie,1965). However the disease does have a seasonal pattern, with most cases occurring from February to September. The subsequent spread and disease epidemics were shown to be associated with heavy infestations of Calomvs callosus. This rodent has a pastoral life-style, and will also readily occupy houses in close association with man. It was shown that 50% of Calomvs callosus caught in one epidemic area were infected with Machupo virus (Johnson et al., 1966). Persistent infections of the virus have also been demonstrated in this rodent. Transmission from Calomys callosus to man is probably by skin abrasions, aerosols or in contaminated food and water (Johnson and Webb, 1975). Rodent control has been extremely effective

15

in containing and terminating epidemics of BHF (Mercado, 1975). Consequently with an effective rodent control programme in place, the number of cases of BHF occurring annually is very low and the geographical spread has been halted.

8. Antigenic relationships.All members of the Arenavirus group have some

antigenic relationship with each other, depending on which serological assay system is used (Howard and Simpson, 1980). The complement fixation and immunofluorescence tests have been frequently used with the result that the viruses naturally form groups relative to their geographical origin (Casals et al.,1975). Using complement fixation, all the New World members are related, with Junin, Machupo, Amapari,Parana, Latino and Tacaribe being closely related. Pichinde and Tamiami are not as closely related to each other or the other members of the Tacaribe complex by this test. Figure 1 shows the geographical locations of the Arenaviruses in the New World group. Of these viruses, Guanarito and Sabia, have only recently been isolated and classified. By complement fixation tests, Guanarito virus was most closely related to Junin, Tacaribe and Amapari viruses (Tesh et al., 1994). Complement fixation and immunofluorescence tests with Sabia virus indicate that it is a new member of the Tacaribe complex (Coimbra et al., 1994). Using the

16

C3

USA

TAMIAMI

TACARIBE

VENEZUELA

GUANARITO

PICHINDECOLOMBIAAMAPARIFLEXALBRAZIL

MACHUPOBOLIVIALATINOSABIA

PARANA

ARGENTINAju n I n

KEY•— VIRUS

• COUNTRY

FIGURE 1 MAP OF SOUTH AMERICA SHOWING THEGEOGRAPHIC DISTRIBUTION OF NEW WORLD ARENAVIRUS ISOLATES (after Tesh et a l . , 1994)

17

indirect immunofluorescence test, LCM and Lassa viruses were closely related and only distantly related to the Tacaribe complex (Rowe et al., 1970b). For this reason, LCM virus is grouped with the Old World viruses. The geographical location of the Old World viruses is shown in Figure 2.

Serological tests, performed using polyclonal antisera, established antigenic relationships between the viruses, but could offer no explanation as to how these geographically isolated African and South American groups of viruses had evolved. In recent years monoclonal antibodies have been used to study these relationships further. It appears that Central African virus isolates [Mobala strains] are antigenically more closely related to virus isolates from Southern Africa [Mopeia strains] than to those isolated from Western Africa [Lassa strains] (Ruo et al., 1991). During the last five years, the nucleotide sequence information of the RNA segments of several arenaviruses has also been published. Comparison of amino acid sequences of the viral proteins has confirmed the serological relationships and phylogenetic trees can be constructed for each viral protein. Further details of these will be given in the molecular biology section (section 1.C.3).

18

Key to Figure 2

Areas of LV isolation

Area of Mobala, Ippy and Sku virus distribution

Area of Mopeia.virus distribution

19

Lassa

MobalaIppySku

Mopeia— '

FIGURE 2 MAP OF AFRICA SHOWING GEOGRAPHICDISTRIBUTION OF OLD WORLD ARENAVIRUS ISOLATES { a f t e r L loyd , 1983)

C. BIOCHEMISTRY AND MOLECULAR BIOLOGY

1. Virion structure and virus replicationMost of the early work on the virion structure of

arenaviruses was done with LCM and Pichinde viruses (Buchmeier and Parekh, 1987). Virus particles are pleomorphic, enveloped, lipid solvent sensitive and range in diameter from 50-300nm (Murphy and Whitfield, 1975). The particles mature by budding through the plasma membrane and contain inclusion bodies indistinguishable from host cell ribosomes. Ribosomes and ribosomal sub­units have been isolated from virus preparations (Pederson and Konigshofer, 1976), so confirming the presence of ribosomes in the virions.

Three structural proteins are major components of the virus particle and a schematic diagram of an arenavirus particle is shown in Figure 3. These proteins are the nucleoprotein (N), with a molecular weight of 60 kilodaltons (kDa), and two glycoproteins, G1 and G2, with molecular weights of 40 and 35 kDa respectively (Ramos et al., 1972). These are approximate sizes for the proteins and vary with different arenaviruses. The nucleoprotein is the most abundant protein, constituting 58-70% of the protein in the virus particle (Vezza et al., 1977) . The glycoproteins are located in the virus membrane as surface projections or spikes, which are 5-lOnm long. Other quantitatively minor proteins have been recovered in purified virus preparations. These include the L

20

OJ * oCLJO

toc ' to

CL(U OJ CD> <u ■ 1 c c LJc<u

Êo

oc_cn.

cu"4— QJ

-4—O

•O too o

uoL_ O

C_V_l3

Q_ JD >N C l n . C

IZ i c c ID Z — I 0 0

TDLOCLCDUOOJV_)3C

FIGURE 3 SCHEMATIC DIAGRAM OF AN ARENAVIRUS PARTICLE(a f te r Bishop, 1990)

21

protein, which is 200 kDa in size, and is thought to be the RNA polymerase (Buchmeier and Parekh, 1987). Also the Z protein, which has been identified as a zinc-binding protein (Salvato and Shimomaye, 1989).

The internal nucleocapsids of the virion are a composite of viral RNA and the N protein. There are two major forms and both are seen as closed circular structures with a "beaded" appearance. These beads may be deposits of N protein and they have been estimated as 3- 4nm in diameter, occurring at approximately 5nm intervals (Young et al., 1981). RNA from the nucleocapsids is linear, single-stranded and sensitive to ribonuclease digestion.

Arenavirus replication is slow in vitro, usually taking 2-3 days to attain maximum virus production (Bishop, 1990). There is insufficient evidence to fully elucidate the exact details of replication, but attachment of the virion to the cell surface is probably by interaction of a surface viral glycoprotein and a host cell receptor. The ribonucleoprotein complex enters the cytoplasm and viral RNA synthesis begins. N and L proteins are produced, followed by the glycoproteins and the Z protein. Although the time scale for protein synthesis will depend on the multiplicity of infection, studies of LCM virus replication have demonstrated the sequence of events in all arenaviruses. N protein can be detected 6-12 hours postinfection, with maximum levels at 48-72 hours post infection (Buchmeier et al., 1978).

22

Glycoproteins are usually detected between 24-48 hours post infection and their synthesis usually declines by 60 hours post infection.

As mentioned earlier, arenaviruses often produce persistent infections in animals and this is also the case in vitro. These persistent infections, whether in vivo or in vitro. are characterized by the production of the N protein, often without G1 and G2 proteins on the cell surface, and lack of production of infectious virus particles (Hotchin et al., 1975). The phenomenon of persistent infection is not yet fully understood, but the development of persistent infection in animals appears to be related to the state of the host's immune system (Rawls et al., 1981). For many of these studies the model system of LCM virus in mice has been used.

2. GenomeNucleic acid extracted from arenavirus preparations

contain two size classes of RNA. These are the large species (L), which is approximately 7.5 kilobases (kb) long, and the small species (S) , approximately 3.5kb long (Clegg, 1992). The L strand encodes for the L and Z proteins, while the S strand encodes for the glycoproteins and the N protein. The arenavirus genome is bi-segmented and single-stranded, with the expression of the two segments being unusual because of the "ambisense" arrangement (Auperin et al., 1984). This means that the open reading frames of the genes are in opposing

23

orientation on the virus strand and virus complementary strand of each RNA segment.

Both the L and S strands are arranged so that the open reading frames are in opposite orientations. The open reading frame for N messenger RNA is at the 3' end of the genome and extends to nucleotide residue 1766, whilst the open reading frame for the glycoprotein is initiated from the 5' end of the S strand (Bishop and Auperin, 1987). Translation for N starts at the 3' end, but translation for the glycoproteins, in the same segment, starts at the 5' end. Studies with LCM, LV and Pichinde viruses show this to be a common strategy for the arenaviruses. Some members of the bunyavirus family also have ambisense RNA (Roizman, 1990). Translation of the proteins from each gene is illustrated in Figure 4.

During the last five years nucleic acid sequence information has been published on many of the arenaviruses. As mentioned in the sub-section on antigenic relationships (section B.8), this has confirmed most of the serological data and further defined the relationships between individual arenaviruses. When the amino acid sequences of the N protein are aligned, a phylogenetic tree can be constructed (Figure 5) illustrating the relationships of arenaviruses to each other (Clegg, 1992). Sequence information has been similarly compared from the precursor glycoproteins (GPC) of arenaviruses and a dendrogram constructed (Figure 6).

24

m

ŸÂ

I m

JD_k:CVJ

I<Zq: i

CLID

•ÏÎ

i

in

04D JZ)_h:m

<zcc(/)

M fin

FIGURE 4 DIAGRAM REPRESENTING TRANSLATION OF THE ARENAVIRUS GENOME. Dotted areas are coding regions for the virus proteins Z, L, G1, G2 and N. Arrows show the direction of translation. ( a fter C[egg,1992)

25

3. Major viral structural proteinsN protein is the most abundant viral protein,

comprising up to 58% of the virion protein mass of an arenavirus (Vezza et al., 1977). It is synthesised early in infection and its synthesis continues after the onset of glycoprotein production. Its molecular weight ranges from 60-68 kDa, depending on the virus, and was accurately determined as 63 kDa for LCM virus by Buchmeier and Parekh (1987). Incorporation of the radioactive amino acids, leucine and methionine, has been reported, but no sulphate or phosphate is utilised (Gard et al., 1977). Attempts to show phosphorylated proteins in arenaviruses have generally been unsuccessful (Buchmeier and Parekh, 1987), however there has been one report that N appeared as a soluble phosphorylated protein of 63 kDa size using LCM virus (Bruns et al.,1986).

Where complete amino acid sequences are available for the N proteins of arenaviruses, alignments and comparisons can be made as in Figure 5. It can be seen that the two strains of LCM virus, WE and Armstrong (Arm), are very closely related, as are the two strains of LV, Sierra Leone and Nigeria. There is more than 90% identity between the two strains of each virus. A comparison of the amino acids of the N proteins of LCM virus and LV, shows approximately 60% homology at the amino acid level. A closer relationship is identified between Lassa and Mopeia viruses, where there is 70-80%

26

onCOc_1z

QJ o QJ LU EfT 3C

C L3 J D > <t m

c ' cZ QJ-£Zu

C L

uro c3 —V

mu L J

_ J

ZL J_1

C Loz:

QJCOQJ— I■r ^

z Lofo m (/) (/) (/) 10 (D (D

FIGURE 5 PHYLOGENETIC TREE SHOWING AMINO ACID SEQUENCE RELATIONSHIPS OF A R E N V IR U S NUCLEOPROTEINS ( a f t e r Clegg, 1992)

27

identity (Wilson and Clegg, 1991). With the New World viruses, Pichinde (Auperin et al., 1984) has the least homology with the rest of the group. Junin (Ghiringhelli et al., 1989), Machupo (Clegg and Lloyd, 1991) and Tacaribe (Franze-Fernandez et al., 1987) viruses share 54-60% homology.

Apart from the N protein, the other two structural viral proteins of arenaviruses that are synthesised in significant quantities are glycoproteins (Figure 4).These two glycoproteins, G1 and G2, are produced from the cleavage of a precursor glycoprotein, GPC, which has an approximate size of 70 kDa. G1 and G2 are synthesised in equimolar quantities in LCM (Burns and Buchmeier, 1991) and Pichinde virions (Vezza et al., 1977), with their sizes having been estimated at 44 kDa for G1 and 35 kDa for G2. The proteolytic cleavage site for GPC in LCM virus has been identified to be within a 9 amino acid sequence, with the probability that the site is the Arg- Arg sequence at amino acids 262-263 (Buchmeier et al.,1987). This region is conserved in Pichinde and LV, so these viruses probably share the same cleavage site (Bishop, 1990) . Tacaribe is an exception in the arenavirus group, because only one glycoprotein has been identified as a structural component of virions (Franze- Fernandez et al., 1987).

Information available on the biochemistry and structure of arenavirus glycoproteins is still sparse.Two recent studies using LCM virus (Wright et al., 1990;

28

QJJDn(DUCD

QJIDC

uCL~ L J

CD

QJ(=LO2:

ONm<IDCD(QV)CD

CDV)OCDtoCOCD

FIGURE 6 DENDROGRAM OF RELATIONSHIPS BETWEEN ARENAVIRUS GLYCOPROTEIN NUCLEOTIDE SEQUENCES (a f t e r Clegg e + a l 1990)

29

Burns and Buchmeier, 1991) have examined the structure and processing of the glycoproteins, but little work has been carried out with the more pathogenic arenaviruses.In LCM virus the sugars that are incorporated into GPC are mannose and glucosamine, whereas G1 contains glucosamine, fucose and galactose. G2 incorporates glucosamine and fucose (Buchmeier et al., 1987). When looking at post-translational processing of the glycoproteins, it was found that cleavage of GPC into G1 and G2 was prevented by treatment of cells with tunicamycin (Wright et al., 1990). Transport of the unglycosylated GPC is blocked and the protein aggregates in the rough endoplasmic reticulum. In virions, G1 is the peripheral protein of the virion spike and G2 is an integral membrane protein, which may also interact with N protein (Burns and Buchmeier, 1991).

By using specific polyclonal and monoclonal antibodies directed against antigenic sites on the virion surface, neutralization epitopes on the glycoproteins (Howard, 1987; Buchmeier et al., 1987) and the antigenic relationship of different arenaviruses have been examined. With LCM virus the neutralization domain was found to be on G1 (Bruns et al., 1983). Similar results have been obtained with Junin and Machupo viruses (Johnson et al., 1973), but with LV and Pichinde viruses neutralization tests have proven difficult to perform (Howard, 1987) .

30

When amino acid sequences of the GPC proteins of the arenaviruses are compared, a similar relationship to comparisons of the N proteins can be seen (Figures 5 and 6). One difference is that Pichinde GPC sequence has closer affinity to the Old World viruses (LCM, Mopeia and LV) than Tacaribe virus GPC sequence (Clegg et al.,1990). Comparison of potential N-linked glycosylation sites can illustrate how conserved or diverse the glycoproteins have become during their evolution. In Table 4, the potential glycosylation sites of G1 and G2 of two of the Old World viruses (Lassa and Mopeia) and one New World virus (Junin), have been compared. The sites in G2 for all three viruses show only slight differences. For Gl, Lassa and Mopeia are very similar, but Junin is different with three of the sites being absent. Other sequence comparisons (Franze-Fernandez et al., 1987; Auperin and McCormick, 1989) have shown that G2 is highly conserved between arenaviruses at the amino acid level while Gl is the most variable. This may be due to the position of Gl being close to the virion surface. The N protein has also been shown to be conserved amongst the arenaviruses (Auperin and McCormick, 1989) .

D. LASSA VIRUS

1. Animal ModelLF has a relatively long incubation period, 7-18

days in humans, and its clinical symptoms are easily

31

confused with many other less incapacitating illnesses. Therefore, it was important to identify a suitable animal model to advance an understanding of the disease. Mice are a common animal model for LCM studies, and one of the first studies with LV used mice (Buckley and Casals,1970). There were discrepancies in results when different inbred strains of mice were used, and subsequent studies have shown that with LV the outcome of infection is dependent on the mouse genotype (Peters, 1984).

The disease caused by LV in guinea pigs is similar to the disease in humans. In an early comparative study of LV in squirrel monkeys, guinea pigs and Mastomys natalensis. it was found that there were similar patterns of viral tropism in guinea pigs and primates (Walker et al., 1975). It was subsequently shown that the pathogenicity of LV in guinea pigs is dependent on both the virus strain and the host strain. In tests using the Josiah strain of LV, it was found that 0.3 plaque forming units (p.f.u.) of virus killed 50% of strain 13 guinea pigs (Jahrling et al., 1982). Using outbred Hartley guinea pigs, that were given between 2,000 and 200,000 p.f.u. of virus, only 30% of the animals died. Viral titres were also higher in the tissues of the strain 13 animals. The appearance of neutralizing antibody was very slow in guinea pigs, as in monkeys and humans. In the U.S.A., the inbred strain 13 guinea pig has been a valuable animal model for pathogenic studies and antiviral drug testing (Peters et al., 1987). However,

32

TABLE 4. COMPARISON OF THE POTENTIAL N-LINKED GLYCOSYLATION SITES IN ARENAVIRUS

GLYCOPROTEINS ILLUSTRATING Gl DIVERSITY

LASSA MOPEIA JUNINGl

aminoacid

site aminoacid

site aminoacid

site

79 NMT 78 NAT89 NNS 88 NNS99 NET 98 NET

109 NTS 108 NTS 91 NKS119 NLS 118 NLS 101 NAS167 NLS 168 NLS 162 NRT224 NTT 222 NTS 174 NTS

G2365 NYS 363 NYS 353 NYT373 NET 371 NDT 361 NET390 NGS 388 NGS 378 NNS395 NET 393 NET 383 NIS

The diversity of the Old and New World arenavirus Gl glycoproteins is illustrated, showing that three of the potential glycosylation sites are missing from Junin viral Gl. In Lassa and Mopeia (Old World) Gl potential sites are very similar. G2 potential glycosylation sites are similar in New and Old World arenaviruses.Key to one letter amino acid symbols :N Asparagine S Serine T Threonine L Leucine A Alanine M Methionine E Glutamic acid

R Arginine K Lysine Y Tyrosine H Histidine I Isoleucine D Aspartic acid G Glycine

33

several LV isolates from human patients have been non- pathogenic in guinea pigs while two of these isolates were lethal for cynomolgus monkeys (Jahrling et al.,1985). Thus demonstrating that primate testing is still necessary.

Studies with different species of non-human primates show variation in their susceptibility to LV.Low doses of LV (110 p.f.u.) kill rhesus, cynomolgus and African green monkeys, but capuchin and squirrel monkeys are resistant to this and higher (610 p.f.u.) doses (Peters et al., 1987). During the first 4 weeks of infection, monkeys shed virus from the nasopharynx and in their urine. Viral assays on visceral tissues from dead monkeys showed significant virus replication, but virus was not detected in samples from the brain stem, cerebellum and spinal cord. All monkeys seroconverted, including the ones that died, even while still viraemic. This implies that early antibody response is notassociated with reduced viraemia nor with recovery fromthe disease.

Some rhesus monkeys survived (Peters et al., 1987),even when they were given higher doses of LV (one millionp.f.u.). The antibody response of these animals was similar to that documented in human LF patients (Jahrling et al., 1985). Anti-viral antibodies were detected at 7- 10 days after infection, reaching a maximum titre by 33 days post-infection. Neutralizing antibodies were not

34

detected until 45 days post-infection. In these studies the Josiah strain of LV was used. From the limited studies with LV on non-human primates, it can be concluded that either rhesus or cynomologus monkeys are the best animal model for antiviral drug and vaccine tests.

2. Antiviral TherapyConvalescent plasma has been used successfully for

treating patients infected with arenavirus haemorrhagic fever illnesses since 1979. When convalescent plasma treatment was given to patients with AHF within the first 8 days of illness, it reduced a mortality rate of 3-15% to less than 1% (Maiztegui et al., 1979). However, when LF patients were treated with Lassa-convalescent plasma there was no significant reduction in mortality (McCormick et al., 1986). Laboratory experiments using LV infected guinea pigs and monkeys showed that high levels of IgG in the plasma were necessary for protection and that IgG therapy may be an effective treatment for LF (Jahrling et al., 1984).

There are few life-threatening viral diseases where an effective antiviral drug is available, but for LF there is one. Ribavirin (Virazole) prevents death in LV patients, especially when administered intravenously during the first six days of illness (McCormick et al.,1986). Loss in efficacy of the drug may have resulted from irreversible tissue damage caused by the disease.

35

Only one side-effect has been reported as a result of ribavirin treatment, namely a few cases of reversible anaemia. However, the side-effect was not severe enough to interrupt treatment.

The mode of action of ribavirin, a nucleoside analogue, is to inhibit virus replication and this had been demonstrated in vitro prior to the clinical trials with LV. Ribavirin has also been shown to be effective, both in vitro and in vivo, against B.H.F. and A.H.F. infections (Stephen et al., 1980). Treatment of these diseases, in animal models, is only partially successful Ribavirin protects against the acute haemorrhagic phase of the infection, but not the late neurological sequelae This may be due to the inability of ribavirin to cross the blood/brain barrier (Ferrara et al., 1981).

3. VaccinesConventional preparations of inactivated or

attenuated virus for use as vaccines against LF have not been successful in providing protection against LV challenge in laboratory animals (Peters et al., 1987). Three different methods have been used to inactivate the virus: formaldehyde, ultraviolet irradiation and gamma irradiation. The resultant preparations failed either to protect against lethal challenge, or reduce the effects of the disease, or increase the time to death. During these tests the animals produced antibodies to LV, as demonstrated by immunofluorescence tests. The

36

arenaviruses Mopeia and Mobala have been used successfully to protect monkeys and guinea pigs against LV challenge (Kiley et al., 1979). However, no information is available on the pathogenicity of these viruses in humans, although there is a report that they cause renal and liver damage in primates (Lange et al., 1985). Evidence that Mopeia virus may cause persistent infections in monkeys (Peters et al., 1987) also indicates there is little possibility that this virus will be tested as a vaccine for humans.

With the failure of these vaccine preparations, recent research has focussed on the application of recombinant DNA technology to produce an effective LV vaccine. Vaccines produced by this research would contain only small non-infectious portions of the virus and so the problems encountered when using whole virus would not occur. Two viruses have been used as vectors for inserts of LV genes ; vaccinia and baculoviruses. The LV nucleoprotein gene and the glycoprotein gene have been used separately and together as inserts into vaccinia (Clegg and Lloyd, 1987; Auperin et al., 1988; Morrison et al., 1990) and all have met with various degrees of success.

4. Studies using vaccinia virus and LV gene recombinantsResults of animal experiments using a vaccinia virus

recombinant to protect against LV were first reported in 1987 (Clegg and Lloyd, 1987). The recombinant was

37

produced by inserting the N gene of LV, the Nigerian strain GA391, into the TK gene of vaccinia virus. In these experiments the vaccinia strain was Lister, which had been widely employed for human vaccination in Europe, and the promoter was the 7.5K promoter. Dunkin-Hartley outbred guinea pigs were vaccinated subcutaneously with 10"' p.f.u. of either the recombinant or wild-type vaccinia virus. Twenty eight days later these guinea pigs were challenged intraperitoneally with 10 TCID50 of LV (Nigerian strain). Various post challenge parameters were monitored, including temperature, weight, viraemia, virus specific antibodies and serum aspartate transferase (AST) levels. No LV disease symptoms were recorded in animals vaccinated with the recombinant in the 28 days after challenge that they were monitored. Of the twelve unvaccinated animals or those receiving wild-type vaccinia virus, seven died in the acute stage of the disease, four were culled when terminally ill and one died when a blood sample was being taken.

Further experiments were reported in 1988 (Auperin et al., 1988), in which a different vaccinia strain and a different LV gene were employed for producing the recombinant. The GPC gene of Josiah strain LV was inserted into the New York Board of Health (NYBH) strain of vaccinia virus. Both sets of experiments used the same promoter and the same vaccinia insertion site and Hartley guinea pigs were the animal model. In the 1988 experiments, 10® p.f.u. of either the wild-type or

vaccinia virus recombinant was given intradermally and challenge was 21 days post-inoculation, with 10 p.f.u. of guinea pig cultured LV Josiah strain. With this construct the protection was not complete. All guinea pigs vaccinated with the recombinant virus developed transient low-grade fevers, starting approximately 8 days post challenge and lasting for 4 days. In comparison, all unprotected animals had high fever which persisted until death. This experiment was continued until 61 days post challenge and surviving animals had no LV in their blood or tissues at the end of the experiment. Although the recombinant vaccinated animals developed antibodies to vaccinia by 19 days post vaccination, no antibodies were detected to LV.

With many viral diseases, it is the surface glycoprotein gene that has provided protection against challenge where vaccinia recombinants have been used as the vaccine (Spriggs et al., 1988; Wachsman et al.,1987). The result with the LV GPC gene was therefore surprising. This prompted a study in which a group of researchers compared the protection to LV from vaccinia recombinants with either N or GPC, under the same conditions (Morrison et al., 1989). Many parameters were similar to the 1988 (Auperin et al., 1988) study and the same GPC vaccinia recombinant was used. The vaccinia strain was NYBH, the LV strain Josiah and the animal model was guinea pigs. In the recombinant virus incorporating the LV N gene, the LV gene was inserted

39

into the vaccinia TK gene. A summary of the survival data at the end of the experiment is shown in Table 5.

In this study, animals became viraemic by 8 days post-challenge and in animals vaccinated with recombinant vaccinia viruses, they had cleared the virus by day 11. The authors concluded that there was no correlation between antibody levels to LV and protection, as some animals with relatively high antibody levels to LV did not survive challenge. Survival rates in animals vaccinated with the N recombinant were better than those vaccinated with the GPC recombinant. Fewer animals that received both the N and GPC recombinants at different inoculation sites were protected. There was a better survival rate in the animals that had been inoculated with wild-type vaccinia than in uninoculated animals. In these experiments animals were challenged at three weeks post-vaccination.

This same group of researchers produced a vaccinia recombinant that contained both the LV N and GPC genes (Morrison et al., 1990). By using a double expression transfer vector they were able to generate one recombinant virus, that contained both LV genes inserted into the TK vaccinia gene. The recombinant was injected intraperitoneally into AJ mice, which were then monitored for antibody levels to vaccinia and LV. After one inoculation no antibodies were detected to LV and after a second inoculation low antibody levels were recorded. No challenge data has been published using this recombinant.

40

TABLE 5. PROTECTION OF GUINEA PIGS AGAINST A CHALLENGE OF lO' pfu WITH RECOMBINANT VACCINIA VIRUSES

(modified from Morrison et al., 1989)

Vaccine % guinea pigs surviving

N/v.v. 94GPC/v.V. 79N/v.v. + GPC/v.V. 58NYBH wild-type 39unvaccinated 14

N LV nucleoproteinv.v. vaccinia virusGPC LV precursor-glycoproteinNYBH New York Board of Health vaccinia virus

strain

41

There has been one published report of a LV challenge of recombinant vaccinia inoculated monkeys (Fisher-Hoch et al., 1989). They used the same vaccinia NYBH/LV GPC construct as previous researchers (Auperin et al., 1988; Morrison et al., 1989). Rhesus monkeys were used in this study as the mortality rate from LV infection is usually 100%. Two monkeys were inoculated with 10 p.f.u. of NYBH wild-type vaccinia and four monkeys were inoculated with the same amount of the recombinant virus. Two monkeys from the recombinant group were challenged 37 days post-vaccination with 10 p.f.u. of LV Josiah strain. The remaining two monkeys from this group were challenged 284 days post-vaccination.

By 12 and 15 days post-challenge the monkeys inoculated with the wild-type vaccinia virus had died from LV infection. All four monkeys inoculated with the recombinant virus became febrile by 7 days post­challenge, but all survived the challenge and were eventually sacrificed. They also developed some symptoms of LF, although the severity of the disease was greatly reduced. For example they all had depressed platelet function, lymphopenia and neutrophilia. So although survival was 100%, protection was insufficient to prevent a low severity infection. Unfortunately no studies have been published using the LV N gene vaccinia virus construct and LV challenge in monkeys.

42

Recombinants have also been made with the LV glycoprotein gene and baculovirus as the virus vector. In one report (Hummel et al., 1992), the recombinant was evaluated as a potential antigen in diagnostic assays.It failed to react with some human serum samples that had been shown positive for LV when other antigens were used. At the present time, there have been no reports about its protection ability in animal studies.

There is the possibility that a vaccine to another arenavirus may protect against LF. Recent laboratory tests with rhesus macaques and a live-attenuated vaccine against A.H.F. have shown that the vaccine provides complete protection without side-effects (McKee et al., 1992: McKee et al., 1993). Subsequently this vaccine. Candid no. 1, has undergone human trials and has been inoculated into approximately 70,000 volunteers in Argentina. The ability of this vaccine to protect animals against challenge by other arenaviruses has yet to be determined.

43

SECTION 2. RECOMBINANT VACCINIA VIRUSES

A. ERADICATION OF SMALLPOX

1. ClassificationThe disease smallpox is caused by variola virus, a

member of the family Poxviridae. Included within this family, in the genus Orthopoxvirus, are the viruses variola, cowpox, monkeypox and vaccinia (Moss, 1990a). Members of this genus have identical morphology, are antigenically related and have similar biological properties. Poxviruses are large double-stranded DNA viruses with virus particles that are just visible by light microscopy. More details of the morphology will be included in the virus replication section (Chapter 1, section 2.B).

All Poxviruses that infect vertebrates have a common nucleoprotein antigen (Woodroofe and Fenner, 1962), a property which can be utilized in identification or classification. Further parameters used to classify these viruses, within a particular genus, are cross-protection in animals and cross neutralization of infectivity in tissue culture. More recently cross-hybridization of genomic DNA from virions has been employed as a preliminary test to identify virus isolates (Buller and Palumbo, 1991).

Variola is the only virus in the Orthopoxvirus genus to be classified as an ACDP hazard group 4 pathogen. No

44

laboratory work has been undertaken for several years in the United Kingdom with variola virus strains. Before the eradication of smallpox in 1977, the natural distribution of variola virus had been world-wide, with man being the natural host. Other viruses in the same genus are classified as ACDP hazard category 2 pathogens and have a more limited distribution. Cowpox, for example, is found mainly in western Europe, where it infects not only cows, but cats (Bennett et al., 1986) and rodents (Marennikova et al., 1978).

2. HistorySmallpox is thought to have been present in China

and India for thousands of years, but it was not accurately described before the year 900 and then by a Persian physician (Benenson, 1989). By the fifteenth century it was endemic in Europe, from where the European colonists spread the disease to the Americas, Africa and finally Australia in the eighteenth century (Fenner,1986). During the seventeenth and eighteenth centuries the disease was greatly feared because of its severity, high fatality rate (15-20%) and dermal scarring in survivors. In many countries there was the additional threat of patients being removed to isolation hospitals; a precaution taken to arrest the spread of the disease.

The connection between variola as the causative agent and smallpox, the disease, was established during the last century. Inclusion bodies of variola and

45

vaccinia virus are basophilically stained in the cytoplasm, so it was possible to distinguish them in tisssue samples using relatively crude techniques. As early as 1886 observations were taking place on the variola and vaccinia micro-organisms (Buist, 1886) and by 1931 the importance of these "elementary bodies" was determined (Ledingham, 1931). In 1953 a subcommittee on poxvirus classification was formed which summarised the characteristics of the poxvirus group (Fenner and Burnet, 1957) and this still constitutes the basis for classification of the group today.

Vaccinia virus was the live attenuated vaccine used to achieve the eradication of smallpox. The exact evolution or origins of vaccinia virus are unknown, although there are several theories, mainly based on the differences in host range of the viruses. Both vaccinia and cowpox viruses have a broad host range, unlike variola for which man is the only host. One theory was that vaccinia was derived from variola and modifications to variola had taken place after passage of the virus in rabbits and cows. This theory was tested by passaging variola over one hundred times through rabbits or calves (Herrlich et al., 1963). No alteration was made to the host range of variola. Similar theories that vaccinia was a derivative of cowpox virus or a hybrid of cowpox and variola have yet to be proven.

Recently the genome of variola virus, strain Bangladesh-1975 (Massung et al., 1993), has been

46

sequenced and the nucleotide sequence compared with vaccinia virus, strain Copenhagen (Goebel et al., 1990). One hundred and fifty proteins were found to be similar in the two viruses, but in thirty seven proteins there were noticeable differences (Massung et al., 1993). In a more recent paper (Massung et al., 1994), the amino acid sequences of the variola strain were compared with the amino acid sequences of other poxviruses, with the aim of identifying virulence factors. Comparisons of single gene functions failed to offer any reason for the range of virulence in the poxvirus strains examined, and it was suggested that a complex interaction of several gene products was responsible. As yet there is no indication as to why variola virus was restricted to humans or why the disease, smallpox, was so severe.

3. PathogenesisWith the onset of smallpox there is an incubation

period of 7 to 17 days, followed by fever with temperatures of 39.5° to 40.5° C. This fever usually lasts for 2 to 4 days and is accompanied by headache, back pain and prostration. Clinical diagnosis, at this stage, is difficult and the disease was often confused with influenza, meningitis or pneumonia. For variola virus the normal route of infection is via the respiratory tract (Downie, 1970). The virus initially causes swellings and capillary dilation in the mucous membranes of the mouth and upper respiratory tract, subsequently infecting cells

47

in the skin. In the skin the spread of virus to the epithelial cells causes the initial rash, which appears first on the face, hands and forearms, then the trunk and lower limbs (Christie, 1969). Characteristic vesicles form with fluid accumulation in the dermis, although the number of lesions vary widely. Approximately ten days after pustule formation, dry hard scabs cover the infected areas. Lesions form in the mouth and pharynx, but no scab formation occurs and virus is produced from these ulcerated areas (Benenson, 1989). Few lesions occur in internal organs and this may be attributed to the raised internal body temperature during fever and the fact that the virus fails to replicate above 38.5° C (Nizamuddin and Dumbell, 1961).

A variation in the severity of illness and case fatalities in different outbreaks of smallpox is thought to be due to different strains of variola causing the outbreak. Variola minor virus outbreaks produced less severe illness with case fatality rates of only 1%, but with haemorrhaging occuring in some patients. With the variola major strain, the illness is as described in the preceeding paragraph, and case fatality rates were approximately 30%. Haemorrhaging with disease caused by variola major was rare (Rao et al., 1968) .

Long term side-effects resulting from smallpox disease include scarring and blindness. Scarring occurs after the sebaceous glands are destroyed, and as these are more numerous on the face, this is where most

48

scarring takes place. Many smallpox patients suffered with conjunctivitis while permanent eye damage, resulting in blindness, occurred less frequently (Benenson, 1989).

4. W.H.O. eradication prograimneIn an attempt to reduce the severity of smallpox, a

practice arose in Asia, in about the tenth century, of people being artificially given the disease, either by the inoculation of pus or intranasal infection. This resulted in a less severe disease although case fatalities still occurred, but life long protection was obtained against smallpox (Fenner, 1986). Edward Jenner in 1796 showed that protection could be provided by pus from a cowpox pustule inoculated into the arm, with side- effects of only a small lesion and no fatalities (Jenner, 1798). This practice of vaccination continued in Europe and by 1900 reduced the number of smallpox cases, however in many countries the disease was still endemic. The availability of freeze-dried vaccine in the 1920s also increased the potency of the vaccine in tropical countries.

By 1959 smallpox had almost disappeared from Europe and North America (Figure 7) and in that year a resolution was passed, by the World Health Organization (W.H.O.) Assembly, to eradicate smallpox infection worldwide. One phase of this programme was planned as mass vaccination, which eventually changed to case identification, then isolation and vaccination of known

49

, _ n , r , , ,n\ 195?;WH0 RESOLUTION ON 1967: INTENSIFIEDAFRICA (47) g lo b a l e r a d ic a t io n ^ I program m e

ASIA (38

\ '\AMERICA(251~---

'V . EU ROPE (30)

OCEANIA (15)T 1--- 1---- 1-- 1--- 1----1---1— I I I I I I I 'I I ri1920 1925 1930 1935 1940 1945 1950 195558 62 66 70 74 78

Years

FIGURE 7 NUMBERS OF COUNTRIES IN WHICH SMALLPOX WAS ENDEMIC BETWEEN 1920 AND 1977 (a f te r Fenner, 1986)

Totals in brackets show countries from each continent supplying data

50

contacts (Foege et al., 1971). The disease continued to persist in Asia and Africa, so the programme was intensified in 1967. This involved a greater collaboration between countries where smallpox was still prevalent, and an increased availability and quality control of the vaccine. From 1977 to 1979 surveillance checks were carried out and certificates issued stating which regions and countries were clear of smallpox. In May 1980, such was the success of the intensified programme, that an agreement was reached to end smallpox vaccination, except to laboratory workers handling orthopoxviruses.

Eradication of smallpox was achieved when the risk of infection or disease, in the absence of vaccination or any other control measures, had been eliminated (Evans, 1989). Several factors contributed to the success of the eradication programme: some were connected with the properties of the virus and the disease, others involved social and political factors (Table 6). With many viruses causing infections in Man there is at least one animal host in which the virus can persist or multiply, for example the arenaviruses have a rodent reservoir. This was not true with variola virus, and if the disease could be eliminated from the human population, reinfection would not occur from an animal source.

There was effective protection provided by the vaccine (Table 6). Using vaccinia virus as the vaccine, complete protection from smallpox infection was expected

51

TABLE 6. FACTORS AFFECTING THE ERADICATION OF SMALLPOX

1. Man was only host: no animal reservoir.2. Effective vaccine available; long term

immunity.3. Relatively easy diagnosis.4. Few subclinical cases, no carriers and no

recurrence of disease.5. Only one serotype of variola virus.6. Financial rewards: due to elimination of costs

of illness, quarantine and vaccination.7. Political agreement that the severity and

mortality rates were sufficiently high to justify the costs of eradication.

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for 5 years (Dixon, 1962) with protection from death by the disease for 20 years. With the increased temperature stability of the freeze-dried vaccine and the stabilityof the vaccinia genome, there was no necessity at that time to further improve the vaccine. The quality of the vaccine, for the eradication programme, was assured by two W.H.O. reference centres; one at Connaught Laboratories in Toronto, Canada and the second at the Rijks Institute in the Netherlands (Benenson, 1989) .

During the first 10-12 days after infection a smallpox patient is non-infectious, and not until the distinctive rash appears, does the patient become infectious. Transmission of variola virus is often by person to person contact, so early diagnosis assists in limiting the spread of the disease (Table 6). Eradication was also made easier because there were few subclinical cases of the disease, no carriers and rarely recurrent infections. The financial rewards in eliminating smallpox were significant (Evans, 1986) , coupled with the political and social factors motivated by the severity of the disease and its high mortality rate.

5. Variola virus eradicationAfter the removal of smallpox, the disease, from the

world, there arose the question of whether laboratory stocks of variola virus should be completely destroyed. Under a W.H.O. recommendation, all variola virus stocks have either been destroyed or transferred to one of two

53

laboratories: Centers for Disease Control in Atlanta, U.S.A. or the Institute for Viral Preparations in Moscow, Russia (Joklik et al., 1993). There was also a W.H.O. recommendation to destroy all virus stocks in December 1993, after sequencing the genomes of two strains of variola major and one strain of variola minor. With the completion of these tasks, a debate has developed in the scientific community as to whether the virus stocks should be destroyed. As yet this question is unresolved. Some scientists are advocating further research into the restricted host range of the virus and the structure and functions of the viral proteins (Joklik et al., 1993) . In contrast other scientists are proposing the alternative view, that whole virus is unnecessary for any further research. They advocate that sufficient information could be obtained from available clones and plasmids, or from studies with other poxviruses (Mahy et al., 1993).

B . VIRUS REPLICATION

1. Morpholocrv and virion structurePoxvirions are oval or brick-shaped, approximately

200-400 nanometers long, having axial ratios of 1.2 to 1.7 (Buller and Palumbo, 1991). When viewed under the electron microscope, the external surface of the virions show ridges that are sometimes arranged in parallel rows (Peters, 1956a). Thin sections show the structure of purified virions is composed of a core, lateral body,

54

membrane and envelope (Fenner et al., 1989). Vaccinia virus is the prototype of the orthopox genus, so its structure and composition has been reported in detail.The core is formed from nucleoprotein (Peters, 1956b), intertwining DNA fibres. It is biconcave in shape and the lateral bodies are embedded in the concaves. A lipid- protein bilayer surrounds the core (Dales, 1963). The lipid composition of this membrane is principally cholesterol and phospholipids (Stern and Dales, 1974), which is different from host cell membranes. Surrounding all this is the envelope. At least eight polypeptides form the envelope of extracellular vaccinia virus and of which seven are glycoproteins (Payne, 1978).

The main chemical components of vaccinia virions are protein [90%], lipid [5%] and DNA [3.2%] (Zwartouw,1964). Thirty viral polypeptides have been identified from poxviruses by one-dimensional polyacrylamide gel electrophoresis (Sarov and Joklik, 1972a) and over one hundred on two dimensional gels. When the complete DNA sequence of the Copenhagen strain of vaccinia virus was reported (Goebel et al., 1990), it showed that the genome contained enough DNA, 191,636 base pairs, to encode for 263 proteins greater in size than 65 amino acids.

An unusual characteristic of the poxvirus family is that although they are DNA viruses, they replicate in the cytoplasm of the cell and not in the nucleus. Since poxvirus virions can translate mRNA to make proteins, the virion core contains many viral encoded enzymes which

55

include a DNA-dependent RNA polymerase (Moss, 1991). The poxviruses and African swine fever virus are the only eukaryotic DNA viruses that have their own RNA polymerase (Moss, 1990a). Other viral enzymes, such as capping and methylating enzymes, give the virion enough potential to synthesise translatable messenger RNA. Many of the functions now identified to specific genes are related to virus specified nucleic acid metabolism or synthesis (Johnson et al., 1993).

Poxvirus genomes vary in size from 130,000 base pairs to 300,000 base pairs (Moss, 1990a). All consist of linear double stranded DNA with hairpin loops connecting the two strands at each end (Moss, 1991). DNA restriction mapping revealed a region of Hind III sites in the central region of the genome, which was conserved in the orthopox family (Mackett and Archard, 1979). This region has been used as a reference site when comparing variola virus sequences (Massung et al., 1993) and when establishing relative positions of genes in the genome (Johnson et al., 1993). Although some vaccinia functions have been assigned to specific genes, many remain to be assigned.

2. Virus entry into cellsMost enveloped viruses enter cells by either pH-

independent fusion with the plasma membrane or by a low pH-dependent endosomal route (Moss, 1990a). Poxviruses have two types of virion; the intracellular and

56

extracellular forms, both of which are infectious. Many infectivity studies with vaccinia virus have used the intracellular virions. Electron micrographs (Chang and Metz, 1976) and the use of lysomotropic agents (Dales and Kajioka, 1964) indicate that pH-independent fusion is the most important type of entry by poxvirions. Research is continuing to identify which viral proteins are required for adsorption and penetration (Rodriguez et al., 1987). Reports have shown that the extracellular virions also fuse to the plasma membrane in the same way, although the mechanism is more rapid (Payne and Norrby, 1978; Doms et al., 1990).

After entry into the cell, the virion undergoes a two stage uncoating (Dales, 1965). During the first stage the virion proteins and lipids are removed (Figure 8). There are indications that at least one viral protein, a 23 kDa protein which has trypsin-like activity, may be required for this uncoating (Pedley and Cooper, 1987). In the second stage the DNA of the genome becomes accessible, so that it is susceptible to deoxyribonuclease (Sarov and Joklik, 1972b).

3. Gene expressionAlmost immediately after infection, virus cores are

released into the cell cytoplasm and virus specific messenger RNA (mRNA) is produced. Approximately half the genome is transcribed before DNA replication starts (Moss, 1990b). Two vaccinia virus-specified polypeptides,

57

ceu to en z) o c_ (_ — Q- >

OUW

j= 4-

>. «QJU III!

FIGURES VACCINIA V IR U S REPLICATION CYCLE(a f te r Moss, 1990 a)

58

77 and 82 kDa, have been shown to bind to and initiate transcription from early promoters (Broyles et al.,1988). There is evidence that certain factors of the promoters are conserved between the poxviruses and can function for a heterologous poxvirus. The TK promoter of a fowlpox virus functioned in a vaccinia recombinant virus (Boyle and Coupar, 1986) and conversely a vaccinia virus promoter functioned in a recombinant fowlpox virus (Boyle and Coupar, 1988; Taylor et al., 1988). A viral DNA polymerase, thymidine kinase (TK), thymidylate kinase and a ribonucleotide reductase have been shown to be expressed early in infection (Smith et al., 1989).

With the start of viral DNA replication there is a change from early protein synthesis to late protein synthesis. Pulse-labelling with radioactive amino acids indicates that there may be two sets of late proteins; the ones produced immediately after DNA replication and those that are delayed (Moss and Salzman, 1968). Also, as late protein production starts the synthesis of early proteins stops. Exactly how this is regulated is not fully understood, but the stability of mRNA may be a factor influencing the synthesis of early and late proteins. The stability of mRNA decreases as infection progresses, with the half-life of late mRNA being shorter than that of early mRNA (Sebring and Salzman, 1967) . Many of the viral structural proteins and viral enzymes are made late in infection.

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4. DNA replicationWithin the infactivity cycle, the initiation of DNA

replication varies, in time, amongst poxvirus members. In cells synchronously infected with vaccinia virus, initiation of DNA replication occurs between 1.5 and 6 hours post infection (Salzman, 1960), whereas with fowlpox virus it is 12 to 16 hours post infection (Prideaux and Boyle, 1987). Other factors influencing the onset, are multiplicity of infection and the cell line used for the studies. As poxvirus DNA replication proceeds in the cell cytoplasm, areas of foci of replication can be discerned. These areas, known as viroplasm, appear as granular structures in electron micrographs (Cairns, 1960). The poxvirus genome is so large that as yet the DNA replicating mechanism is still to be deciphered. However at least two enzymes have been identified that are involved in nucleotide metabolism, TK and ribonucleotide reductase, and these are produced in substantial quantities by poxviruses. The TK enzyme was identified as being viral initiated, after its production in a TK" cell line and by physical differences in the enzyme to host cell TK (Kit et al., 1974). Ribonucleotide reductase converts ribonucleotide to DNA precursors and this enzyme is produced in vaccinia virus infected cells soon after infection (Slabaugh et al., 1984). Both vaccinia and fowlpox virus DNA polymerases have been sequenced and shown to have extensive similarities (Binns et al., 1987).

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5. Virion assemblyThe sequence of events leading to the production of

progeny virus is similar in all poxviruses. There are six morphologically distinct stages (de Harvan and Yohn,1966)

1. crescent2. closed immature particle3. closed immature particle with nucleoid4. early core formation within membrane5. mature virion6. mature virion with envelope

The crescents are composed of a bilayer membrane with spicules on the convex surface and electron dense granular material adjacent to the concave surface (Dales and Mosbach, 1968). This viral membrane does not appear to be formed from any host cell membrane (Stern and Dales, 1976), unlike other enveloped viruses, although its precise origin is uncertain. A 65 kDa protein is reported to be a major component of the spicules (Weinrich et al., 1985).

In the immature particle the virus envelope appears circular, enclosing a granular matrix that eventually has a dense nucleoprotein mass. This immature particle lacks many of the mature viral structural proteins, including the major core proteins. As the virion assembly progresses, these major core proteins are produced by proteolytic processing (Silver and Dales, 1982).In the formation of the mature virion, the 65 kDa protein and

61

the spicules on the membrane surface are replaced by 54 and 58 kDa proteins. This process is considered to be important in the change in shape of the immature particle to the brick-shaped mature particle (Essani et al.,1982) .

The whole process of virion assembly can be quite rapid. In vaccinia virus infected cells, progeny virus can be seen under the electron microscope at 4 to 6 hours post infection, with maximum virus yields after 12 to 24 hours. Mature virions are transported from the viroplasm to the cell surface, where they may be enveloped by additional membranes derived from the Golgi apparatus (Dales and Pogo, 1981). Fusion occurs with the cell plasma membrane, and then the virus particles bud from the cell surface. Most poxvirus virions remain intracellular in tissue culture cells at the end of the infection cycle. The amount of extracellular vaccinia virus compared with intracellular virus produced varies from 1 to 30% of progeny virus. This yield is dependent on the virus strain and the cell type used for virus growth. There appears to be no correlation with total virus yield or plaque size (Payne, 1979). There are however antigenic differences between the intracellular and extracellular virus forms (Appleyard et al., 1971).

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C. GENERATION OF RECOMBINANT VACCINIA VIRUSES

ProcedureThe advantages of vaccinia virus as a vector for

foreign genes will be outlined in the next section (section 2D); there are, however, certain properties of the virus that initially make the construction and production of recombinant vaccinia viruses a complex and laborious process. Due to the large size of the vaccinia genome, direct manipulation of the intact genome is impracticable and the viral DNA is non-infectious, so the usual one-step procedure of producing recombinant viruses by infecting cells with viral DNA is impractical (Mackett and Smith, 1986). However the application of marker rescue methodology made the generation of recombinant viruses within vaccinia virus infected cells possible (Smith and Mackett, 1992) .

A further problem is that only poxvirus promoters will function in vaccinia virus (Mackett, 1990), so that when shuttle plasmids for recombinants are being prepared a vaccinia promoter has to be included in the construct. To overcome these difficulties a two-stage procedure has been formulated to make vaccinia recombinants (Figure 9). In the first stage a shuttle plasmid is constructed that contains an E. coli origin of replication, a vaccinia promoter and the foreign gene of interest, which is flanked by vaccinia virus DNA sequences. These sequences are necessary to target the foreign gene into a specific

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STAGE 1 : A recombinant plasmid is constructed, in which the foreign gene is flanked by vaccinia sequences

STAGE 2 : In this stage, infected vaccinia virus mammalian cells are made permeable, to enable the plasmid to incorporate into the vaccinia genome as a virus is replicating

Recombinantplasmid

Vacciniavirus

Transfection

I Ihomologous

recombinationnucleus

. foreign gene incorporated into vaccina genome

cytoplasm

Infectious vacciniarecombinants

A selection is then made of the recombinants, so tha t the non-recombinant vaccinia virus is elim inated. The recombinants can then be grown for the required usage.

FIGURE 9 GENERAL PROCEDURE FOR PRODUCING VACCINIAVIRUS RECOMBINANTS

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non-essential vaccinia gene for insertion into the vaccinia genome (Hruby, 1990).

The second stage in making the recombinants is accomplished by homologous recombination between vaccinia virus DNA and the plasmid DNA. Mammalian tisssue culture cells are infected with a low multiplicity of vaccinia virus and transfected with plasmid DNA (Figure 9). After recombination, less than 1% of the progeny virus will contain the foreign DNA (Moss, 1991), so it is necessary to select these from the wild-type vaccinia virus. There are now many selection methods available and these will be outlined in the next section.

2. Recombinant selectionRecombination occurs between the vaccinia DNA

flanking sequences in the plasmid and the homologous DNA sequences of the vaccinia genome. Vaccinia virus and its recombinants form discrete plaques in cell monolayers. Therefore plaque assays are used to grow progeny virus for plaques to be picked, distinguishable from the wild- type virus either by a distinct phenotype or a selection mechanism that prevents the growth of wild-type virus (Mahr and Payne, 1992). One of the first genes in the vaccinia genome to be used for insertion of foreign DNA was the thymidine kinase (TK) gene. This gene is still frequently employed for insertions (Mackett et al.,1982). When insertions are made into the TK gene, recombinants can be selected by growing progeny virus in

65

TK" cells in media containing 5-bromodeoxyuridine (BUdR).More recently, advances have been made in the

plasmid constructs, so that dominant selectable markers or markers that can provide visual screening make recombinant selection easier (Mackett, 1990). 6- galactosidase (6-gal) reacts with 5-bromo-4-chloro-3- indoyl-6-D-galactosidase (X-gal) to produce a distinct blue colour. By constructing a plasmid that expressed 6- gal and incorporating X-gal into the agarose overlay, recombinants appear as blue areas and can be easily distinguished. Plasmid pSCll was one of the first plasmids to be constructed with this modification for use in recombinant vaccinia virus studies (Chakrabarti et al., 1985).

Plasmids have also been prepared so that recombinant selection can be achieved by growing progeny virus in cells with medium containing mycophenolic acid (Falkner and Moss, 1988). Selection by insertion in the vaccinia host range gene, KIL, has also been achieved (Drillien et al., 1981). Advances are also being made to eliminate the selective marker from the final recombinant virus (Falkner and Moss, 1990), which is especially advantageous if a recombinant is intended for use as a vaccine. Finally, after selection, recombinants are analysed by an immunological detection method or a DNA hybridization method.

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3. Factors affecting expressionThere are three major factors that influence the

quantity of protein expression by foreign genes from recombinant vaccinia viruses. These are the vaccinia virus promoter used in the preparation of the plasmid construct, the site of insertion in the vaccinia genome and the strain of vaccinia virus used to generate the recombinant virus. Various vaccinia promoters have been used in the construction of vaccinia virus recombinants (Moss, 1991). One report, using the same foreign gene, compared four different vaccinia promoters and two different insertion sites (Lyons et al., 1990). The results agreed with those of other researchers, in that late promoters produced the highest levels of protein; the disadvantage of late promoter products was that they were often not immunogenic. Consequently the choice of promoter will depend on the final use of the recombinant virus (Smith and Mackett, 1992). Many successful constructs have used the vaccinia 7.5K promoter and the vaccinia TK gene for insertion (Brochier et al., 1988; Wertz et al., 1987). Also, special plasmid vectors have been constructed to permit rapid cloning and expression of foreign genes in vaccinia. Improvements to these vectors are continuing (Fuerst et al., 1989; Davison and Moss, 1990).

Studies with vaccinia virus deletion mutants have shown that there are a large number of non-essential regions in the genome which are not essential for virus

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growth (Boursnell et al., 1988; Perkus et al., 1989) . To date, relatively few of these insertion sites have been used to produce successful recombinants. Sites into which insertions have been made include the vaccinia virus TK, growth factor, ribonucleotide reductase and fusion protein genes. Recombinant viruses made from these insertions have attenuated in vivo replication, but are still able to replicate in cell culture; useful attributes for vaccine production (Hruby, 1990). It is also possible to make insertions into more than one gene and have proteins expressed from these insertions by one vaccinia recombinant (Perkus et al., 1985).

The third main factor influencing levels of expression, is the strain of vaccinia virus employed to make the recombinants. WR strain is the most common laboratory strain used. This strain was derived from the New York City Board of Health (NYBH) vaccine strain, after passaging in mouse brain (Fenner et al., 1988) . Details and comparisons of recombinants made with attenuated vaccinia strains are given in section 2D2. Two research projects illustrate how successful recombinants for vaccine use were made with the WR strain, but how recombinants made with less virulent strains failed to protect animals. A WR strain recombinant with Epstein- Barr virus glycoprotein protected cotton top tamarins against Epstein-Barr virus induced lymphoma, whereas a Wyeth strain recombinant virus failed to protect (Morgan et al., 1988). Similarly, a WR recombinant with Bacillus

68

anthracis protective antigen partially protected animals against spore challenge, whereas a Connaught strain recombinant virus did not protect (lacono-Connors et al.,1991). Both the Wyeth and Connaught vaccinia strains were derived from the NYCBH strain, and had been used in human vaccination against smallpox.

4. Uses of recombinant vaccinia virusesRecombinant vaccinia viruses have been used

successfully in three major research areas. These areas are for basic research in protein biochemistry and immunology, as recombinant vaccines, and as immunotherapy vectors. In protein biochemistry, recombinant vaccinia viruses have been used to express microbial structural proteins, enzymes and peptides (Smith et al., 1987;Thomas et al., 1988a; Thomas et al., 1988b). Proteins expressed by vaccinia recombinants mostly undergo the same post-translational modifications as they would in their natural environment (Rice et al., 1985) and are normally expressed as biologically active components. Consequently recombinant vaccinia viruses can be used to study the structure-function relationship of individual proteins, peptides or enzymes. For the production of large quantities of protein a plasmid vector which also incorporates the T7 RNA polymerase promoter within the the vaccinia recombinant can be utilized (Barrett et al.,1989). Another use of vaccinia recombinants is for determining which proteins induce an immune response in

69

animals. Again individual proteins can be analysed and vaccinia recombinants have been utilized extensively to study the immune response to human immunodeficiency virus proteins (Klavinskis et al., 1989).

In experimental animals, proteins expressed by vaccinia recombinants have beeen used to reduce or clear malignant tumors. One such study involved the use of a recombinant virus expressing a polyoma virus tumor antigen (Lathe et al., 1987). Studies involving a protein associated with malignant melanoma, p97, showed that mice injected with a recombinant expressing this protein rejected cell implants of p97 presented in the abnormal malignant inducing form (Estin et al., 1988). Rats were also shown to be protected against tumor formation by a recombinant vaccinia virus expressing a modified protein associated with human breast tumors (Hareuveni et al.,1990). These animal experiments demonstrate that recombinant vaccinia viruses have applications in immunotherapy. The potential of recombinant vaccinia viruses as vaccines will be outlined in the next section (2.D).

D. VACCINIA AS A VECTOR

1. HistorySince 1980 vaccinia virus has no longer been

required as a vaccine for smallpox (section 2. A4), however this was not necessarily a demise for vaccinia

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virus, as studies with this and other orthopoxviruses were being encouraged by W.H.O. (Moss, 1991). About this time, advances in genetic engineering were applied to the vaccinia genome, making it possible to use vaccinia as a vector for foreign genes. Subsequently it was found that many advantages of the vaccinia vaccine were retained by recombinant vaccinia viruses, which then had potential as new vaccines (Fenner et al., 1988). Some of these advantages are listed in Table 7, and they include ease of administration, temperature stability, and genome stability. Large inserts of DNA, estimated up to 25Kb (Smith and Moss, 1983), can be made into the vaccinia genome without affecting virus replication. This means that as well as large single inserts, several genes can be inserted and expressed simultaneously in one vaccinia recombinant making multivalent vaccines a possibility (Perkus et al., 1985).

As vaccinia virus had been used to safely vaccinate humans effectively against smallpox for many years, the virus strains had been extensively tested in humans (Hruby, 1990). There were a few, rare complications, such as post-vaccinial encephalitis, and these will be considered in the next paragraph. Vaccinia virus and its recombinants stimulate cell-mediated and antibody responses in experimental animals (Mackett, 1990) and humans (Zagury et al., 1987). Therefore, one reason that the foreign proteins expressed by vaccinia recombinants have often been immunogenic, is that these proteins

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TABLE 7. ADVANTAGES AND DISADVANTAGES OF VACCINIA VIRUS AS A POTENTIAL VACCINE VECTOR.

ADVANTAGES

1. Good temperature stability.2. Genome very stable.3. Administration by single scratch.4. Large inserts of DNA can be introduced.5. Multiple inserts of DNA are possible.6. Extensive use in humans as smallpox vaccine.7. Stimulates cell-mediated and humoral immunity.8. Correct processing and presentation of

antigen to the immune system.

DISADVANTAGES

1. Medical complications to vaccinia vaccination.2. The biological effects of insertions into many

vaccinia genes is insufficiently documented.

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undergo the same post-translational modifications that would occur during their production in their natural state (Moss and Flexner, 1987).

2. Attenuation of vaccinia virusAlthough the advantages for using recombinant

vaccines outnumber the disadvantages, the latter cannot be ignored (Table 7). Even though vaccinia virus was used extensively world-wide for vaccinating humans, the side- effects from vaccination may prove an obstacle for licencing recombinant vaccinia virus vaccines. Only certain strains of vaccinia virus were licenced for human inoculation by W.H.O. Those strains were Lister, EM-63 from Moscow and the New York Board of Health (NYBH) strain in the U.S.A. (W.H.O., 1972). There was a correlation between the adverse side-effects and the vaccinia strain used for inoculation. For example, post- vaccinial encephalitis was reported to be as frequent as 1 case in every 4,000 in Holland when the Copenhagen and Bern strains were employed (Polak, 1973). The frequency of this complication was greatly reduced when only the Lister strain was used (Polak et al., 1963). Lister was the most common vaccine strain in Europe and Asia (Benenson, 1989). A progressive vaccinia virus complication was also reported in people with impaired immune systems (Fenner et al., 1988), so the increased incidence of acquired immunodeficiency syndrome (AIDS), in subsequent years, could add to this problem.

73

Now that many vaccinia genes have been assigned functions, it is possible to attenuate vaccinia virus for use as a vaccine vector, by deleting or inactivating virulence or non-essential genes from the vaccinia genome (Cox et al., 1992). Deletion of the vaccinia virus growth factor gene produced recombinant viruses that had decreased virulence in mice and rabbits (Duller et al., 1988). A further possible area for deletion in the vaccinia genome would be genes encoding host range determinants (Perkus et al., 1990; Drillien et al.,1981). To date there have not been any reports of recombinants made with this type of vector.

Most recombinants have been made with the Western Reserve (WR) strain, because this strain induces high expression levels of foreign genes. However this strain will never be used in humans as it produces neurovirulent side-effects in mice. Research is on-going to produce recombinants with attenuated vaccinia strains that retain immunogenic potential and could possibly be used as human vaccines. Two examples of these attenuated strains are NYVAC and LC16m8 (Tartaglia et al., 1992; Watanabe et al., 1989). The NYVAC strain was produced by deleting genes associated with virulence and recombinant viruses have been generated which express pseudorabies and Japanese encephalitis virus genes (Brockmeier et al.,1993; Konishi et al., 1992). These recombinant viruses have undergone successful trials in pigs. LC16m8 is a temperature sensitive attenuated strain derived from the

74

Lister strain, which was inoculated into approximately 10,000 people in Japan without any reported complications (Hashizume et al., 1985). Recombinant viruses have been generated that express the human T-cell leukemia virus type 1 glycoprotein gene. Neurovirulence tests have also been carried out in rabbits comparing the Lister and WR strains (Shida et al., 1988). These showed that WR and its recombinants were the most neurovirulent; Lister and its recombinants less neurovirulent, with LC16m8 and its recombinants the least neurovirulent. As studies continue to assign functions to genes in the vaccinia genome (Johnson et al., 1993) and virulence factors are identified, improvements for the safe use and application of vaccinia as a vector should be possible.

More recent research with the vaccinia virus strain LC16m8 and a similar attenuated strain, LClGmO, has shown that these strains retain their attenuation when used as vectors for foreign genes (Sugimoto and Yamanouchi,1994).In an attempt to identify the optimum conditions for immunogenicity from recombinants made with these vaccinia virus strains, a comparison was made using different foreign genes inserted into different insertion sites in the vaccinia genome. The best immunogenicity was obtained with the LC16mO strain and the haemagglutinin gene as the insertion site. A potential new vaccine for rinderpest virus has been made with the LC16mO strain incorporating the rinderpest virus haemagglutinin gene (Yamanouchi et al., 1993). Using this recombinant

75

vaccinia virus 100% of cattle were protected from rinderpest virus challenge.

E. AVIAN POXVIRUSES AS VECTORS

Avian poxviruses have a restricted host range and large genomes ranging in size from 240 to 300 kb (Boursnell, 1992). These factors recommend them as recombinant virus vectors, especially since sequence information is now available on the genomes of these viruses. The efficacy of recombinants made with fowlpox virus have principally been examined in birds (Boyle and Coupar, 1988; Bayliss et al., 1991), with the notable exception of a study in mice (Wild et al., 1990). By using a fowlpox virus recombinant expressing measles virus fusion protein, mice were protected against challenge with measles virus.

With the productive replication of avipoxviruses being restricted to avian species (Paoletti et al.,1994), there had been speculation that recombinants made with these viruses would not produce immunity in non- avian species. Although avipoxviruses do not replicate in mammals, it has now been demonstrated that they do express foreign genes and some products from the avipoxvirus genome (Taylor et al., 1994). Foreign genes expressed have induced humoral and cellular immunity and provide protection in a range of animals (Cadoz et al., 1992; Tartaglia et al., 1993), These studies have used an

76

attenuated licensed poxvirus vaccine for canaries (ALVAC) as the vector and this virus and its recombinants have been demonstrated safe in newborn and immunocompromised mice (Paoletti et al., 1994).

Phase 1 human vaccine trials have also been completed with an ALVAC recombinant incorporating the rabies virus glycoprotein gene (Taylor et al., 1994). A comparison was made with the current inactivated rabies virus vaccine. All volunteers receiving the ALVAC recombinant virus produced neutralizing antibodies to rabies virus, with these antibody levels being elevated by a booster inoculation. The vaccine was administered subcutaneously, with only a mild transient reaction at the inoculation site. These studies with ALVAC as the vector demonstrate the potential of poxvirus recombinant vaccines made with this virus in animals and humans, and should encourage research aimed at expressing other foreign genes.

F. HOST IMMUNE RESPONSE TO VACCINATION

Many investigators have studied the effect of anti­poxvirus antibody on poxvirus infectivity in vitro and in vivo. obtaining differing results. In one report, neutralizing antiserum titres to vaccinia virus in rabbits were found to vary with the route of inoculation (McNeill, 1965). The highest titres were obtained following intradermal inoculation and the lowest from

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intramiiscular ihocül^tibn7 Neutralizing antibody titres to vaccinia virus in man follow the pattern of a long­term memory response (Duller and Palumbo, 1991). Titres could still be detected in human serum 10 years after vaccination (McCarthy et al., 1958) and revaccination increased the neutralization antibody titre, even if the primary vaccination had been 20 years earlier (Cutchins et al., 1960) .

Although some workers reported that animals had antibodies to vaccinia virus (Watanabe et al., 1989), rarely are these results confirmed in publications with recombinant vaccinia viruses. Once again different inoculation routes are often used so antibody levels may vary considerably. An antibody response to vaccinia in an animal does show that the animal has responded to the recombinant vaccinia virus vaccine. A synopsis on antibodies to poxviruses indicates that specific antibody to poxviruses is not important in recovery from infection, but it is important in preventing reinfection (Duller and Palumbo, 1991).

Cytotoxic T cells (CTL) have been demonstrated in response to infection with vaccinia virus in mice, rats and sheep (Andrew et al., 1989; Issekutz, 1984). The response also correlates with recovery from disease. CTL have not been demonstrated in primates after poxvirus infection and in primates the recovery from disease seems to be associated with natural killer cells (NK).Cytolytic activity in leukocytes, due to NK cells, after

78

vaccinia, iiifëct:ibrT has been shown in rhesus monkeys and humans (Stitz et al., 1984; Moller-Larsen, 1979). This complex situation of how a host's immune system responds to poxvirus infections is not easy to interpret, especially when the same virus can cause different diseases in different hosts. An example of this is the the disease caused by cowpox virus. Cowpox in humans and cows causes a localized pustule skin lesion: in cats a generalized infection with pustular rashes occurs.

From the many studies now being undertaken to assign functions to genes in the vaccinia genome, it is emerging that poxviruses have certain in-built manoeuvres which help them to evade the host's immune defenses (Johnson et al., 1993). Most of the functions thus far assigned have been identified from the similarity of their amino acid sequences to known proteins that have characteristic functions. These functions are then verified for the corresponding biological activity. One such protein, a secreted protein, identified and assigned to the C3L vaccinia gene, blocks the complement cascade pathway of a host (Kotwal et al., 1990). Other proteins, possibly as many as three, counteract the effect of interferons (Chang et al., 1992; Beattie et al., 1991).

In both vaccinia and cowpox genomes, a secreted glycoprotein that binds to interleukin-1 (IL-1) has been identified (Smith and Chan, 1991; Alcami and Smith,1992).IL-1 is a cytokine that is produced in response to infection and tissue damage, and is directly involved in

79

the 7:ytbkine^mediated host immune response. A difference between the Copenhagen and WR vaccinia strains has been identified, as cells infected with the Copenhagen strain do not secrete this glycoprotein (Alcami and Smith,1992). Therefore, as more information becomes available in assigning functions to vaccinia genes, it will become easier to understand the interaction of at least one poxvirus with at least one host.

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SECTION 3. AIMS OF THESIS

To date little has been reported on recombinant vaccinia viruses expressing LV proteins. The overall aim of this thesis was to investigate the use of recombinant vaccinia viruses as a vaccine to protect against LV infection. This was achieved by studying the following:

1. Generation and characterization of recombinant vaccinia viruses expressing LV glycoproteins with the WR and Lister strains of vaccinia virus.

2. Comparison of the ability of two cell lines to produce large quantities of the recombinant vaccinia viruses for purification before inoculation into animals.

3. Determination if recombinant vaccinia viruses would protect guinea pigs from a lethal challenge of LV. This included comparison of the protective efficacy of recombinant vaccinia viruses expressing either the glycoproteins or the nucleoprotein of LV. A number of parameters of LV disease were examined in vaccinated and unvaccinated guinea pigs.

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CHAPTER 2

MATERIALS AND METHODS

MATERIALS AND METHODS

A. CELL CULTURE, VIRUSES AND ANTISERA

1. CellsMonkey kidney (CVl) cells and rabbit kidney (RK13)

cells were grown in Eagle's modified minimal essential medium (EMEM) supplemented with 10% foetal calf serum (ICN laboratories), Glutamine (2mM), penicillin (lOOU/ml) , streptomycin (100/xg/ml) and N-2- hydroxyethylpiperazine-N-2-ethanesulphonic acid (HEPES) buffer (lOmM, pH 7.5) were also added to the medium. Similar medium was used for cultures of TK"143 cells, except an extra supplement of 5-bromodeoxyuridine (BUdR) at 25/zg/ml was added (Pensiero et al., 1988).

2. VirusesStocks of Western Reserve (WR) and Lister strains of

vaccinia virus were grown in CVl cells. The WR strain of virus was initially supplied by Dr. M. Mackett (Paterson Institute for Cancer Research, Manchester) and the Lister strain obtained from the Swiss Serum and Vaccine Institute, Berne, suppliers of vaccine for human inoculation. Lassa virus strain GA 391 was of Nigerian origin and supplied by Dr. G. Lloyd (C.A.M.R., Porton Down). This strain of Lassa virus was originally isolated at Porton Down, from a patient in Zaria, northern Nigeria

82

(Grundy et al., 1980) in 1977 and has been subsequently grown and passaged three times in Vero E6 cells.

3. Antisera and monoclonal antibodiesHeat-inactivated antisera to vaccinia virus was

prepared by inoculating guinea pigs with purified vaccinia virus. Animals were bled at 8 weeks post­inoculation. Heat-inactivated LV antisera was from human LV infected patients and supplied by Dr. David Brown, Colindale. For LV studies within the ACDP level 4 laboratories at CAMR a monoclonal antibody to LV, monoclone 47, prepared by Dr. Graham Lloyd was used. Monoclonal antibodies specific for LV glycoproteins G1 and G2, were prepared and supplied by Dr. George French, The Salk Institute, Swiftwater, U.S.A.

B . INFECTIVITY TITRATIONS

Vaccinia virus stocks were frozen and thawed three times to release virus particles from any cellular material. Plaque titration assays were done in either CVl or RK13 cells. Confluent monolayers of cells were seeded and allowed to grow overnight in 12 well plates at 37°C. Virus dilutions were made in Leibovitz (L15) medium containing 0.1% bovine serum albumin (B.S.A.). lOOjil of 10-fold dilutions of virus stock was added per well.After virus adsorption, 1.5 ml of EMEM containing 2% foetal calf serum was added and the plates incubated for

83

72 hours. The cells were then fixed with 10% formal saline and stained with 1.5% crystal violet. Virus plaques were counted after the plates had been rinsed and dried.

C. TRANSFECTION AND SELECTION OF RECOMBINANT VIRUSES

1. Transfection of cellsA pSCll recombinant plasmid containing the LV GPC

gene was generously provided by Dr. J.C.S. Clegg (Clegg and Lloyd, 1991). Recombinant vaccinia virus was prepared by transfecting subconfluent vaccinia virus infected CVl monolayers, with the plasmid, by the calcium phosphate precipitation method (Mackett et al., 1985). Cells were grown overnight to 80% confluency and infected at a multiplicity of infection (m.o.i.) of 0.1 p.f.u. per cell of either WR or Lister strain of vaccinia virus. Five hours post infection, 5jag of calcium phosphate precipitated DNA from the LV GPC pSCll plasmid was added to the infected cells, and incubation continued for a further 40 hours. Progeny virus was harvested, frozen and thawed three times before being used for plaque assays.

2. Selection of recombinantsTK' recombinants were isolated by plaquing in human

TK‘ 143 cells. For these assays a solid overlay of 1% low- melting point agarose (SeaPlaque, FMC), containing 25jng/ml of BUdR and 300/ig/ml of X-Gal (Sigma) was used

84

(Chakrabarti et al., 1985). Recombinant virus plaques stain blue by this procedure and can be easily identified. Blue plaques were plaque purified three times (Mackett and Smith, 1986). Expression of the Lassa glycoproteins was tested by immunofluorescence with human antisera specific for LV (supplied by Dr. D. Brown, PHLS, Colindale).

D. INDIRECT IMMUNOFLUORESCENCE

1. Permeabilized cellsCVl cells were grown to 90% confluency on glass

slides and infected with either wild-type vaccinia virus or recombinant vaccinia viruses. At twenty four hours post infection, the slides were washed three times in phosphate buffered saline (P.B.S.), then immediately fixed in acetone at -20°C for 15 minutes (Pensiero et al., 1988). The slides were allowed to air-dry, after which they were stored at -20®C until required. Slides were rehydrated in P.B.S. containing 0.1% B.S.A. for 15 minutes at room temperature. Excess P.B.S. was removed from the slide and antisera at an appropriate dilution added. LV antisera from human patients (section A.3) had to be pre-absorbed with vaccinia infected CVl cells, to overcome extremely intense non-specific background fluorescence.

After incubating the cells with the antisera for one hour and washing three times in P.B.S., goat anti-human

85

immunoglobulin G (IgG) fluorescein isothiocyanate (F.I.T.C.) conjugate (Sigma) was added for one hour.After washing to remove unbound conjugate, the slides were mounted in 50% glycerol/saline and viewed under a fluorescence microscope.

2 . Non-permeabilized cellsThis method was used to examine for any

immunofluorescence on the surface of cells. CVl cells were grown on slides and infected as described above. After washing with P.B.S., instead of fixing in acetone, the cells on slides were immersed in a freshly prepared solution of 2% formaldehyde/P.B.S. for 5 minutes (Rice et al., 1985). Washings, treatment with antisera, followed by goat anti-human F.I.T.C. conjugate and viewing were the same procedures as described above for permeabilized cells.

E. RADIOLABELLING AND POLYACRYLAMIDE GEL ELECTROPHORESIS

Monolayers of CVl cells were infected with wild-type or recombinant vaccinia virus at a m.o.i. of 15 for 24 hrs. at 37°C. The medium was removed, the cells washed with P.B.S. and incubated in methionine-free media for 1 hr. Intracellular viral proteins were pulse-labelled with 50/xCi of [ S] methionine (lOOOCi/ml) in methionine deficient medium for 3 hrs (Rice et al., 1985). Medium was removed and the cell monolayer washed three times

86

with ice-cold P.B.S. Cells were lysed by scraping into 100/zl of RIPA buffer (1% sodium deoxycholate, 1% Triton X-100, 0.2% sodium dodecyl sulphate [S.D.S.], 150mM NaCl, 50mM Tris hydrochloride [pH 7.4]). A further 100/zl of RIPA buffer containing 2% Triton X-100 was added and the lysate kept on ice for 15 minutes. Cell debris was removed by centrifugation at 10,000 r.p.m. for 10 minutes. The supernatant was removed to a fresh tube and 20^1 of antisera or monoclonal antibody added (Payne,1992) . Following incubation at 4°C for 1 hr., lOOjLtl of 10% (w/vj Protein A-Sepharose that had been pre-washed with RIPA buffer and pre-absorbed in P.B.S. was added. The samples were incubated and continually mixed by rotation for Ihr. at 4°C (Lyons et al., 1990). Protein A-Sepharose beads were centrifuged at 10,000 r.p.m. for 5 minutes and the pellets washed three times in ice-cold RIPA buffer without Triton X. Immune complexes bound to the beads were released by adding IBOfil of S.D.S. polyacrylamide gel electrophoresis sample buffer and heating to 100°C for 5 minutes. Samples were electrophoresed in 10% discontinuous S.D.S. polyacrylamide gels (Laemmli, 1970).

F. PURIFICATION OF VIRUS FOR INOCULATION OF ANIMALS

To avoid potential cross-reaction in the guinea pigs to the cellular material in which virus stocks were grown, all vaccinia viruses, whether wild-type or recombinant, were partially purified before inoculation.

87

virus was grown in RK13 cells and released from the cells by three cycles of freezing and thawing, followed by homogenization. Cell debris was removed by low-speed centrifugation for 10 minutes (Tartaglia et al., 1992). The supernatant was then layered over a 36% sucrose cushion (sucrose in ImM Tris pH 9.0) and centrifuged at 70,000g for 60 minutes. Virus pellets were resuspended in sterile P.B.S., assayed by plague titration and the virus suspension stored at -70°C.

G. ACDP LEVEL 4 LABORATORIES

1. Virus laboratoryLV is an ADCP hazard group 4 virus (Table 1), so all

manipulations with infectious material were carried out at ACDP containment level 4. Training, expertise and supervision were provided by Dr. Graham Lloyd and the facilities were at the Centre for Applied Microbiology Research, Porton Down. The unit comprises a laboratory (Figure 10), which is entered through an airlock from a changing room with shower. Materials only leave the laboratory through a double-sided autoclave. All rooms in the unit are maintained under negative pressure, with the input and exhaust air passing through High Efficiency Particulate Air (HEPA) filters. Access to the laboratory is via the autoclave annex to the changing room, where everyday clothing is exchanged for protective clothing.

88

FIGURE 10. ACDP LEVEL 4 CONTAINMENT LABORATORY AT CAMR, PORTON DOWN.

89

n

;m

When in the laboratory, two pairs of gloves are worn; a thin latex pair under a pair of white linen gloves.

Within the laboratory are a series of inter­connecting safety cabinets, operating under negative pressure. Back-up fans and stand-by generators, ensure that negative pressure will be maintained in the event of system failures. Material is passed into the cabinets through the high security double-sided autoclave. Passage of material from one cabinet to another is through inter­connecting portholes. Integrated within the cabinets are a low speed centrifuge (Denley), a refrigerator, -20°C freezer and 37°C incubator. Also included are an inverted microscope (Leitz), a fluorescence microscope (Leitz) and an ELISA analyser (Anthos 2000) . Each cabinet is decontaminated with formaldehyde vapour at the end of each working day.

2. ACDP level 4 animal facilitiesAnimal experiments, involving infectious LV and

subsequent analysis of animal samples (haematology, virology and biochemistry), was carried out under ACDP containment level 4 laboratory conditions. The laboratories comprise a suite of rooms that were accessed through an airlock and shower. The rooms are kept under negative pressure and a large double-ended autoclave was used for the decontamination of infectious material. All input and extracted air is double HEPA filtered, and effluent chemically treated before release. Personal

90

protective clothing included a full-face biological respirator. All clothing is removed and discarded on exiting the laboratory, and personnel shower out.

H. ANIMAL STUDIES

I. Guinea pigsOutbred Dunkin-Hartley strain female guinea pigs,

weighing 250-350g., were obtained from Harlan-Porcellus, Bicester, Oxford. The animals were kept and monitored in accordance with the Home Office Scientific Procedures Act 1986 (Home Office, 1989). Daily recordings of rectal temperature and body weight were made, as well as daily food and water intake. Serum samples, obtained by cardiac puncture at various times post-vaccination and post­challenge, were tested for antibody levels to vaccinia and Lassa viruses. Blood samples, obtained post­challenge, were also used for biochemical, haematological and virological studies at ACDP containment level 4 conditions.

2. Vaccination and challengeGroups of guinea pigs were prepared for

scarification with wild-type vaccinia or recombinant vaccinia viruses, by shaving a small area on their hind quarter to expose the skin. The area was cleaned with sterile P.B.S. and between 50-100^1 of virus applied. Shallow scratches were made on the skin surface to ensure

91

virus penetration into the dermis. Each animal received a dose of 1 X 10® p.f.u. of virus. Some animals were given a booster inoculation, with the same amount of virus, three weeks after the first scarification. Challenge was 1 x 1Q2.5 tciDso of LV strain GA391 by the intraperitoneal route.

3. Haematology and BiochemistryBlood samples, obtained by cardiac puncture, were

collected in ethylenedinitrilotetraacetic acid (E.D.T.A.) and heparin tubes and analysed using a Cell-Dyn 900 Hematology Analyzer (Sequoia-Turner Corporation, California). Isotonic buffered saline (pH 7.2) was used as a diluent and a haemoglobin reagent (Rapid Lyse,Abbott) for lysing red blood cells. Samples were kept at 4°C and analysis carried out within 2 hours. The following values were measured: white cell, red cell and platelet counts. Differential white cell examination was carried out on methanol-fixed samples, stained with Hema 'Gurr rapid staining kit (B.D.H.).

With LV infections high aspartate aminotransferase (AST) levels are often associated with fatalities (Chapter 1, section B.5), therefore AST levels were also measured in the guinea pig sera. AST and alanine aminotransferase (ALT) levels in the guinea pig sera were measured using a Photometer 4020 System (Hitachi) with Boehringer Mannheim reagents.

92

4. ELISAa . Vaccinia virus

Sera antibody levels to vaccinia virus were determined using an ELISA. Antigen for coating plates was prepared by infecting CVl cells at a low m.o.i. for 24 hours with wild-type virus. Infected cell sheets were washed several times in P.B.S., scraped into P.B.S., freeze/thawed then centrifuged at 750gr for 5 minutes to remove cell debris (Morgan et al., 1988). The resulting lysate was diluted to a final concentration of 5-10/xg of protein per ml and used to coat Immuno plates (Nunc). Uninfected cells were processed the same way and both plates tested with positive and negative sera. Assays were performed by first blocking excess binding sites with 1% skimmed milk powder in P.B.S. Dilutions of serum were made in 1% skimmed milk in P.B.S., added to the washed plate and incubated for 1 hr. at 37°C (Lyons et al., 1990). Wells were washed in 0.05% Tween 20 in P.B.S. (PBST) and goat anti-guinea pig IgG peroxidase conjugate (Jackson Immunoresearch Laboratories), diluted 1 in 1000 was added. Following a further incubation at 37°C for 1 hr., the wells were washed with PBST before addition of 2,2-Azino-bis(3-ethybenzoline-6-sulphonic acid) [ABTS] peroxidase substrate. The colour was allowed to develop for 1 hr. at 37°C, before the reaction was stopped with O.IM citric acid. Readings were taken on an ELISA plate reader at 405nm.

93

b. LVSera antibody levels to LV were determined by ELISA

Purified LV strain GA 391 was used to coat 96 well plates. Serum samples were diluted and incubated at 37°C in an incubator/shaker for 20 minutes, after which time the wells were washed in PBST. A further 20 minute incubation was carried out after the addition of goat anti-guinea pig horseradish peroxidase (Sigma) and the plates washed in PBST. The substrate 3,3', 5,5'- tetramethylbenzidine in phosphate/citric acid.buffer (Sigma) was added. The reaction was stopped after 20 minutes with 2M sulphuric acid and readings taken at 450nm on an Anthos 2000 spectrophotometer.

5. Virus studiesVero cells were grown to confluency in 96 well

plates and dilutions of the serum samples added. After 7- 10 days incubation at 37°C the cells were fixed with formaldehyde/saline at 4°C overnight. Any antigen was detected by a specific anti-LV monoclonal antibody (47), reacting with anti-mouse IgG horseradish peroxidase and using tetramethylbenzidine as the substrate.

94

CHAPTER 3

RESULTS

SECTION 1. IN VITRO STUDIES

A. INTRODUCTION

The LV GPC gene had been inserted into the pSCll plasmid by Dr. Chris Clegg (CAMR) and was then transfected, at CEDE, into vaccinia virus infected cells. Two different strains of vaccinia virus. Lister and WR were used (Chapter 2 section C.l). With the completion of three cycles of plaque purification in TK' 143 cells, it was necessary to determine if the recombinant vaccinia viruses, WRGPC and LISGPC, were expressing LV glycoproteins that would react with LV antisera. The method used in these tests is described in Chapter 2 section D.l. The CVl cell line was employed and anti­human LV antisera kindly supplied by Dr. David Brown,PHLS Colindale (Chapter 2 section A.3). The antisera was diluted 1:100 for the analysis and wild-type vaccinia virus infected cells were used as a control.Permeabilized and non-permeabilized cells were examined for internal and cell surface fluorescence respectively.

A further method employed for analysing the proteins expressed by the recombinant vaccinia viruses in vitro was radio immune precipitation. The radio-isotope, [®®S] - methionine, was used to radiolabel infected cells, then cell lysates were immunoprecipitated with anti-LV human serum (Chapter 2 section E).

95

For the initial characterization of the proteins produced by the recombinant vaccinia viruses, anti-LV human antiserum was the only LV serum available. At a later date, monoclonal antibodies specific for each of the LV glycoproteins (G1 and G2) became available from the Salk Institute. The reaction of these monoclonal antibodies in permeabilized cells infected with the recombinant vaccinia viruses, was studied by indirect immunofluorescence. After a positive reaction was obtained with these monoclonal antibodies, their cross­reactivity was tested against other arenavirus strains from the virus collection at CAMR.

Prior to in vivo work, it was anticipated that it would be necessary to grow relatively large stocks of vaccinia virus or recombinant vaccinia viruses, in cell culture for purification and inoculation into animals. Both CVl and RK13 cells have been used to grow vaccinia or recombinant vaccinia viruses for animal studies (Wantanabe et al., 1989; Shida et al., 1988). Consequently, a series of experiments was undertaken to optimize and standardize the conditions for virus growth in cell culture prior to any animal work.

96

B . RESULTS

1. Expression of viral crivcoproteins inside infected cells

Cells infected with WRGPC showed bright fluoresence (Figure 11), whereas there was no fluorescence in the control WR vaccinia virus infected cells (Figure 12). A similar result was obtained when the LISGPC (Figure 13) and control LIS vaccinia virus infected cells (Figure 14), were compared, however, the intensity of the fluorescence was slightly less in the LISGPC infected cells when compared with the WRGPC infected cells.

The reaction of permeabilized WRGPC and LISGPC infected cells with the monoclonal antibodies (MAb) to LV glycoproteins G1 (MAb number 52-134-23) and G2 (MAb number 52-135-17) was also examined (Chapter 2 section A.3). A dilution of 1:50 of these MAbs gave the optimum fluorescence. There was less reaction with the LV G1 MAb (52-134-23), when compared with the LV G2 MAb (52-135- 17), from both recombinant vaccinia virus infected cells. A punctate fluorescence similar to that seen with the human LV antisera (Figure 11) was observed in the reaction of the MAbs with both the WRGPC and LISGPC infected cells. There was no detectable fluorescence with the wild-type vaccinia virus infected cells.

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FIGURE 11. INDIRECT IMMUNOFLUORESCENCE OF WRGPC INFECTED, ACETONE FIXED CELLS.

Antibody used for this reaction was anti-LV human serum.

FIGURE 12. INDIRECT IMMUNOFLUORESCENCE OF WR INFECTED, ACETONE FIXED CELLS.

Antibody used for this reaction was anti-LV human serum.

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FIGURE 13. INDIRECT IMMUNOFLUORESCENCE OF LISGPC INFECTED, ACETONE FIXED CELLS.Anti-LV human serum (1:100) was the antibody used for analysis.

FIGURE 14. INDIRECT IMMUNOFLUORESCENCE OF LIS INFECTED, ACETONE FIXED CELLS.Anti-LV human serum (1:100) was the antibody used for analysis.

99

2. Expression of viral crlvcoproteins on the surface of infected cells.

There was fluorescence on the surface of the non­fixed WRGPC infected cells (Figure 15) but no detectable fluorescence on control WR infected cells (Figure 16). A similar pattern of surface fluorescence to WRGPC was observed with LISGPC infected cells (Figure 17) . No fluorescence was discernible on the control LIS infected cells (Figure 18).

3. Radiolabellina of viral glycoproteinsThe two glycoproteins that form part of the LV

structure are G1 and G2, and these have molecular weights of approximately 45 and 38 kDa respectively. During virus replication these glycoproteins are cleaved from a precursor glycoprotein, GPC, which has a molecular weight of approximately 80 kDa. To demonstrate that the recombinant vaccinia viruses were expressing these glycoproteins in vitro, infected cells were radiolabelled with [®®S] -methionine and the cell lysates immunoprecipitated with anti-LV human serum. The result of separating the products of the immunoprecipitated material from WRGPC and WR infected cells on 10% S.D.S. polyacrylamide gels is shown in Figure 19. Polypeptides of approximate molecular weight corresponding to the LV glycoproteins are prominent in the WRGPC lysate material. Other polypeptides were immunoprecipitated by the anti-LV human serum, none of which appear to correspond with the

100

FIGURE 15. INDIRECT IMMUNOFLUORESCENCE OF WRGPC INFECTED, NON-FIXED CELLS.

Antibody used for this reaction was anti-LV human serum.

FIGURE 16. INDIRECT IMMUNOFLUORESCENCE OF WR INFECTED, NON-FIXED CELLS.

Antibody used for this reaction was anti-LV human serum.

101

ri

W

Mw*PA;Â':.%WA

FIGURE 17. INDIRECT IMMUNOFLUORESCENCE OF LISGPC INFECTED, NON-FIXED CELLS.Anti-LV human serum (1:100) was the antibody used for analysis.

FIGURE 18. INDIRECT IMMUNOFLORESCENCE OF LIS INFECTED, NON-FIXED CELLS.Anti-LV human serum (1:100) was the antibody used for analysis.

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* ' T

*’7-*

i ' A

three proteins in the WRGPC lysates. A protein equivalent in size to LV G2 was resolved as a double band on the S.D.S. gel, which is characteristic of the LV G2 glycoprotein.

4. Reactivity of monoclonal antibodies to Lassa virus qlvcoproteins with Arenavirus strains.

Permeabilized virus infected Vero cells were used for these studies, which were performed in the ACDP containment level 4 laboratories. Arenavirus isolates examined from the Old World group were Lassa (strains GA391 and PA), Mopeia (strains 801152, 801150 and 801148) and Mobala. New World arenavirus isolates used were Tacaribe, Junin, Machupo and Pichinde. The MAbs to LV glycoproteins, G1 (52-134-23) and G2 (52-135-17) were used at 1:50 dilution.

None of the New World arenaviruses cross-reacted with these MAbs (Table 8). There was also no reaction with the Mobala virus isolate from Central Africa and similarly no reaction with two of the Mopeia strains (801150 and 801148). However, one Mopeia virus strain (801152) did react with both MAbs, giving a moderate intensity of fluorescence (Table 8). Both LV isolates, one from Nigeria (GA 391) and one from Sierra Leone (PA) , gave good immunofluorescence reactions with the G1 and G2 MAbs.

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FIGURE 1 9 . LV GLYCOPROTEINS LABELLED WITH [^^S ]-M E TH IO N IN E AND IMMUNOPRECIPITATED WITH HUMAN A N T I-L V SERUM

-130

G PC — — 000 — 75G 1 - — E ^ 50

G 2 — ***■ “ 39— - 27

- 1 7

B

Lane A . CVl cells infected with WRGPC, radiolabelled with [ S] -methionine and immunoprecipitated with anti-LV human serum.

Lan e B. CVl cells infected with WR vaccinia virus, radioiabeiied with [ S] -methionine and immunoprecipitated with anti-LV serum.

GPC Precursor glycoproteinG1 cleaved glycoproteinsG2

Numbers indicate the size of molecular weight markers in kiiodaitons (BioRad).

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TABLE 8. REACTION OF ARENAVIRUSES WITH MONOCLONAL ANTIBODIES (MAbs) SPECIFIC FOR LV

GLYCOPROTEINS, G1 AND G2.

Virus (strain) anti-Gl MAb anti-G2 MAbOLD WORLDLassa (GA 391) + + + +Lassa (PA Sierra

Leone)+ + + +

Mopeia (801152) + +Mopeia (801150) - -

Mopeia (801148) - -

Mobala (CAR) - -

NEW WORLDTacaribe (TRVL

11573)- -

Junin(Espandola)

- -

Machupo (AA2 8 8 1 1 1)

- -

Pichinde (Stam47 63/1)

- -

KEY : ( ) virus strainno reaction

+ moderate fluorescence ++ fluorescence

105

5. Growth of recombinant viruses in different cell linesVirus infectivity assays were carried out as

described in Chapter 2 section B and details of the sources of the vaccinia virus strains are outlined in Chapter 2 section A.2. Prior to these studies vaccinia virus (Lister strain) had been passaged twice in CVl cells and Western Reserve (WR) strain had been passaged once in Vero cells. The recombinant vaccinia virus,LISGPC, had received three plaque purifications in TK“143 cells and had been passaged once in CVl cells..Recombinant vaccinia virus, WRGPC, had received three plaque purifications in TK'143 cells and had been passaged twice in CVl cells.

Table 9 shows that there was no difference in the virus infectivity titres when stocks of wild-type vaccinia or recombinant vaccinia viruses were assayed in either CVl or RK13 cells. It had been noted, in routine laboratory work, that better yields of wild-type vaccinia virus were obtained after virus growth in RK13 cells. To establish whether or not this would also occur with the recombinant viruses, and how many passages would be needed to achieve an increased yield, one wild-type virus and two recombinant viruses were passaged once in RK13 cells. A m.o.i. of 1 was used and all viruses were harvested at the same time, namely 3 days post infection. Virus samples were assayed for infectivity in both CVl and RK13 cells to determine if there is any difference

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TABLE 9. INFECTIVITY TITRES OF VACCINIA AND RECOMBINANTVACCINIA VIRUSES IN CVl AND RK13 CELL LINES.

virus logio infectivity titre (p.f.u./ml)CVl RK13

Lister 6.3 [0.12] 6.5 [0.08]WR 7.0 [0.16] 7.3 [0.18]LISGPC 6.4 [0.05] 6.1 [0.13]WRGPC 6.2 [0.07] 6.6 [0.11]

Figures in brackets [ ] denote standard error.

107

in using a heterologous cell line for virus titrations.As shown in Table 10, one passage in RK13 cells was

sufficient to increase the infectivity titres of both wild-type vaccinia virus and the recombinant vaccinia viruses. Increases of 1-2 logs were obtained after one passage with both recombinant vaccinia viruses while smaller increases were obtained with wild-type virus. Also,following one passage in RK13 cells virus infectivity titres were higher if assayed in RK13 cells compared with the CVl cell line. Finally, comparison of virus yields of wild-type vaccinia virus after one passage in either CVl or RK13 cells showed no apparent difference in infectivity titres (Table 11).

In conclusion higher virus titres and yields of infectious virus were obtained with both wild type strains when grown in RK13 cells, and similar infectivity titres were obtained when assayed in either CVl or RK13 cells.

C. DISCUSSION

There was good, intense immunofluorescence in permeabilized cells infected with either WRGPC or LISGPC viruses (Figures 11 and 13). The antisera used was human antisera from a Lassa fever convalescent patient and not specifically for the LV glycoproteins. It was also necessary to pre-adsorb the antisera before use, because

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TABLE 10. INFECTIVITY TITRES OF VACCINIA AND RECOMBINANTVACCINIA VIRUSES AFTER ONE PASSAGE IN RK13CELLS.

virus logic infectivity titre (p.f.u./ml)CVl

beforepassage

afterpassage

RK13before after passage passage

WR 7.0[0.03] 7.4 [0.06] 7.3[0.07] 8.2[0.12]LISGPC 6.4 [0.15] 7.7 [0.14] 6.1[0.11] 8.4[0.09]WRGPC 6.2[0.09] 7.5 [0.13] 6.610.03] 8.3[0.11]

Figures in brackets [ ] denote standard error.RK13 cells were infected at a multiplicity of 1, incubated at 37°C and harvested after 72 hours.

109

TABLE 11. INFECTIVITY TITRES OF VACCINIA VIRUSES AFTERPASSAGE IN EITHER CVl OR RK13 CELLS.

virus logic infectivity titre (p.f.u./ml)Grown CVl

plagued plagued CVl RK13

Grown RK13plagued plagued

CVl RK13WR 5.6[0.05] 5.4[0.04] 7.9 [0.15] 8.2 [0.16]Lister 4.7[0.08] 5.2[0.12] 7.2[0.03] 7.5[0.07]

Figures in brackets [ ] denote standard error.Either CVl or RK13 cells were infected at a multiplicity of infection of 1, incubated at 37°C and harvested 72 hours after infection.

110

of a high non-specific background. Perhaps fewer problems may have been encountered with the serum if the rabbit cell line RK13 had been used in these experiments instead of the monkey cell line CVl.

The LV glycoproteins were expressed on the surface of cells infected with either WRGPC or LISGPC viruses (Figures 15 and 17). Authentic arenavirus glycoproteins, G1 and G2, are transported to the cell surface (Buchmeier and Parekh, 1987), although there is disagreement as to whether the precursor glycoprotein, GPC, is found on the cell surface or it is retained inside the cell (Buchmeier and Parekh, 1987). From the cell surface immunofluorescence preparations with WRGPC and LISGPC viruses, it appears that some LV glycoproteins produced by these recombinant vaccinia viruses are being transported to the cell surface (Figures 15 and 17).

Proteins of corresponding molecular weights to the authentic LV glycoproteins were immunoprecipitated from infected cell lysates of WRGPC (Figure 19). A similar result was obtained with LISGPC (results not shown) and corresponding proteins were not detected in WR or Lister vaccinia virus infected cells. A more ideal situation would be to either use a collection of MAbs specific for the LV glycoproteins for immunoprécipitation or to resolve authentic radiolabelled LV glycoproteins on the same S.D.S. polyacrylamide gel. Other researchers have had problems radiolabelling and immunoprecipitating the LV glycoproteins from recombinant vaccinia viruses

111

(Auperin et al., 1988; Morrison et al., 1989). Western analysis was also unsuccessful with the anti-LV serum they used and moderately successful Western transfers were obtained with a collection of specific MAbs. These recombinant vaccinia viruses had been demonstrated to be partially protective in vivo (Auperin et al., 1988).

The MAbs 52-134-23 and 52-135-17 cross-reacted with virus isolates of LV from Sierra Leone and Nigeria and with one Mopeia virus isolate (reference strain 801152). The Mopeia virus strain that did cross-react was a particularly virulent strain in guinea pigs (Lloyd,1983) . Similar results were obtained by researchers at C.D.C., Atlanta, with these MAbs and their collection of arenavirus isolates (Ruo et al., 1991). One notable difference was that their ten Nigerian LV isolates failed to react with anti-Gl MAb 52-134-23, whereas in this study the same MAb reacted with Nigerian LV isolate GA 391- This result would suggest antigenic variation amongst Nigerian isolates of LV.

When a comparison was made of virus yields from CVl and RK13 cells, for both the wild type vaccinia viruses and recombinant vaccinia viruses, better yields of infectious virus were obtained after growing in RK13 cells. Consequently, all viruses were grown and plagued in RK13 cells when required for animal experiments.

112

SECTION 2 : ANIMAL STUDIES

A. INTRODUCTION

1. Experimental designThe guinea pigs were divided into five groups of

eight animals per group. They were treated as follows.One group was scarified with WR wild-type virus, and three other groups with the recombinant vaccinia viruses either WRGPC, or LISGPC or LISN (i.e. a Lister strain of vaccinia virus expressing LV nucleoprotein and provided by Dr. C. Clegg). Each guinea pig was scarified with 1 x 10® p.f.u. of the appropriate virus (Chapter 2 sectionH.2). A positive control group had PBS scarifications prior to challenge with LV.

Four weeks after the initial scarification, half the animals in each group were given a second scarification with 1 X 10® p.f.u. of the appropriate virus. Eight weeks after the initial vaccination all animals were challenged with 1 X 10 '® TCIDso of LV by the intraperitoneal route (Chapter 2 section H.2). Guinea pigs were monitored for 35 days post-challenge, after which time the experiment was terminated. During the experimental period, if any guinea pig was observed to be under distress it was culled.

113

B . RESULTS

1. Post-immunisation status of animalsBy three days post-immunisation, the shallow

scratches that were made on the skin of the animals, to allow the virus better penetration, had disappeared from those guinea pigs treated with vaccinia virus. By seven days post-scarification, small raised inflamed areas, 5- 10mm. in diameter, had appeared at the site of scarification. After nine days, raised areas on some guinea pigs had increased in size to 15mm. and lesions appeared. By day eleven, all lesions had started to heal and scabs began forming. Animals scarified with wild-type virus had larger areas of lesion formation, with larger scabs, than animals scarified with recombinant vaccinia viruses.

The lesion and scab formation were all very localized to the area of scarification and there was no apparent spread of virus from these localized areas on the skin. When scab formation was complete, the scabs dried and hardened and were quickly shed, along with a small area of fur. A distinct bald patch of skin was visible when the scabs were lost, but new fur rapidly covered this area. There was no apparent difference in the food and water intake of the guinea pigs scarified with virus, in comparison with the P.B.S. scarified guinea pigs. Temperature and weight patterns were also

114

the same and apart from the discomfort at scarification, the animals showed no adverse affects.

2. Antibody levels to vaccinia virus and LVNo serum anti-vaccinia antibodies were detected in

the control (PBS) guinea pigs (Table 12). All animals immunized with either recombinant vaccinia virus or wild- type vaccinia virus had detectable serum anti-vaccinia antibodies at four weeks post-immunization. By eight weeks post-immunization, prior to LV challenge, there were slight increases in the anti-vaccinia antibody levels. Similar serum antibody levels were detected in guinea pigs that received a booster inoculation at five weeks post-immunization (data not shown).

Prior to challenge with LV, serum anti-LV antibodies were undetectable in any animal by ELISA.

3. Post-challenge status of animalsThe disease LF in humans displays a variable pattern

of symptoms and responses (Chapter 1 section B.5), that are reproducible in guinea pigs. The incubation period is a few days, followed by a pyrexia ranging from 38°C-41°C, lasting for several days. Guinea pigs have frequently been used as the animal model for LV and it has been shown that there are varying degrees of pathogenicity, dependent on the combination of LV and host strains (Chapter 1 section D.l). For this reason a disease profile could not be pre-written, although inclusion of

115

TABLE 12. SERUM IgG ANTIBODY LEVELS TO VACCINIA VIRUS

Vaccine Time post­vaccination(weeks)

Mean absorbance(405nm) *

WRGPC 4 weeks 0.51 [0.09]8 weeks 0.60 [0.07]

LISGPC 4 weeks 0.46 [0.08]8 weeks 0.53 [0.04]

LISN 4 weeks 0.51 [0.07]8 weeks 0.55 [0.06]

WR 4 weeks 0.54 [0.09]8 weeks 0.60 [0.10]

PBS 4 weeks 0.05 [0.04]8 weeks 0.07 [0.03]

* The values for pre-immune sera were subtracted from these values.Numbers in brackets [ ] represent standard error of the mean.

116

the PBS positive control guinea pigs did confirm a typical LF disease profile in challenged animals. Therefore, for each response studied, a direct comparison could be made between the recombinant vaccinia virus groups and the LV infected animals. Data presented in the following sections is the mean of the eight guinea pigs from each animal group.

4. Animals surviving LV challengeIn the unvaccinated animals (PBS), six animals died

leaving two surviving animals. The same number died in the WR inoculated group (Table 13). Most deaths occurred between days 10 and 15 post challenge (p.c.). Data presented after this time are the means of the surviving animals. Two deaths occurred in the group of WRGPC inoculated animals and only one death in the group of LISN inoculated animals. All animals in the LISGPC inoculated group survived challenge. There was no difference in the average survival time of animals administered with either recombinant vaccinia virus or PBS.

5. WeightFor 6 days following virus challenge, the weight of

the unvaccinated animals (PBS) remained static (Figure 20). In the table below Figure 20, all weight values are taken to the nearest whole number for display purposes. More accurate weights are shown in the table opposite the

117

TABLE 13. ANIMALS SURVIVING LV CHALLENGE

vaccine no.survivors/ total no. challenged

% survivors mean day of death (p.c.)

WRGPC 6/8 75 13.4 [1.3]LISGPC 8/8 100 -

LISN 7/8 87.5 11.0 [1.8]WR 2/8 25 12.5 [1.4]PBS 2/8 25 12.8 [1.2]

Guinea pigs were inoculated with vaccine and challenged at day 56 post-inoculation with 1 x 10^® TCIDgo LV by the intraperitoneal route.p.c. post-challenge.Numbers in brackets [] represent standard error of the mean.

118

figure). At 8 days p.c. there was an mean average weight increase of 23.7g (from 918.3 to 942g), followed by a dramatic weight reduction of 132g (from 942 to 810g) over the next ten days (8-18 days p.c.). During this period 75% of this group of animals died. The weight of the surviving guinea pigs increased gradually by an average of 42.5g (from 810 to 852.5g) from 18-22 days p.c. In the WR inoculated group, anorexia became evident between days 10 and 14 p.c., with an average weight loss of 69g (from 913 to 844g).

A small weight increase of 0.8g (812.5-813.3g) was observed from 0 to 22 days p.c. in the recombinant vaccinia virus WRGPC inoculated animals. From 12-14 days p.c. there was a 20g (from 797.5 to 777.5g) weight loss, which was rapidly regained. In contrast the LISGPC inoculated animals showed a 47.5g (from 868.7 to 821.2g) weight loss between days 10 and 16 p.c., with the weight having been regained by 22 days p.c. The weights of the LISN inoculated guinea pigs remained constant from 0-10 days p.c., thereafter gaining considerable weight of 104g (from 901 to 1005g) per animal by day 22 p.c.

Anorexia amongst the recombinant vaccinia virus inoculated animals was not evident between 8-10 days p.c. as compared with the other control animals. LISN animals showed no significant alteration in weight during the first 10 days p.c., with no signs of distress and no outward signs such as alterations in food and water intake during this period.

119

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120

Fig.20 BODY WEIGHT OF VACCINATED GUINEA PIGS FOLLOWING CHALLENGE WITH LV

1100

1050

1000

950

900

850

800

750

BODY WEIGHT (gms)

SURVVORS

0 8 10 12 14 16 18 20 22WRGPCLISGPCLISNWRPBS

813874905905912

810863895875910

806865895890908

803875893895918

803871895888942

803869901913897

798856954898877

778828978844850

788821977848843

797830988848810

808848100211876843

813858005880853

DAYS POST INFECTION

— WRGPC LISGPC LISN

WR PBS

6. TemperatureNormal range: 38.8-39.2°CThere was evidence of pyrexia in all the

unvaccinated (PBS) animals, showing a gradual increase to a maximum at 10 days p.c. (Figure 21). A rapid fall in temperature was observed after 10 days p.c. in these animals, where many deaths occurred at day 14 p.c. After 14 days p.c., temperatures slowly returned to near normal at 22 days p.c. WR inoculated animals had pyrexia to 12 days p.c., followed by a rapid decrease in temperature (from 40.7 to 37.7®C) from 12 to 16 days p.c. A similar pattern to the WR wild-type inoculated animals was seen in the LISGPC animals, without the fall to below normal levels.

With WRGPC guinea pigs, there was evidence of a more gradual increase in temperature, with a maximum at day 14 p.c. (40.6°C) and gradually returning to normal at 22 days p.c. LISN inoculated guinea pigs had the least temperature variation (from 38.6 to 39.6°C). A transient temperature rise occurred at 8 days p.c., after which time there was an observed return to pre-challenge temperatures.

After LV challenge, a febrile illness was evident in all groups of animals between 0 and 20 days p.c. In the PBS group, there was a rapid decrease in temperature at 10 days p.c. which continued to day 14 p.c. By 16 days p.c. all the animals had returned to pre-challenge temperatures, except the WR and WRGPC inoculated animals.

121

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122

FIG.21 RECTAL TEMPERATURE OF VACCINATED ANIMALS CHALLENGED WITH LV

RECTAL TEMPERATURE fC)(mean values)

SURVVORS

0 2 4 6 8 10 12 14 16 18 20 22

WRGPC 39.1 38.8 39.4 39.5 39.8 40.5 40.6 40.6 40.2 39.6 39.5 38.6LISGPC 39 39.1 38.8 39.5 40 40.1 40.4 40.6 39 39 38.6 38.7LISN 39 39.3 39 39 39.6 39 39.1 39 39 38.9 38.9 38.6WR 38.8 39 39.6 39.9 40 40.2 40.7 37.9 37.7 38.7 38.5 38.7PBS 38.8 38.8 39 39.8 40 40.3 39.2 37.1 37.7 38.1 38.2 38.3

DAYS POST INFECTION

— WRGPC - + - LISGPC LISN

-a- WR PBS

* HORIZONTAL BARS INDICATE NORMAL RANGE OF VALUES

Only a mild pyrexia was evident throughout the course of the study in the LISN inoculated group of guinea pigs.

7. HaematologyDuring LV infections in humans and guinea pigs,

change in haematological patterns have been documented throughout the course of the disease. Human cases of LF show an acute phase lymphopenia, late neutrophilia and abnormal platelet function (McCormick, 1990). Inbred strain 13 guinea pigs challenged with LV (Josiah strain) had profound lymphopenia, neutrophilia and mild leukopenia (Peters et al., 1987). In this current study haematological patterns were monitored in animals to determine whether or not the guinea pigs inoculated with recombinant vaccinia viruses experienced the same haematological changes throughout infection as LV challenged control guinea pigs. For each parameter, the normal range quoted is that of these groups of animals pre-challenge.

a. PlateletsNormal range: 700-820 x 10*/1The challenge of unvaccinated animals initiated a

severe thrombocytopenia by 7 days p.c., which continued in these and surviving animals until 14 days p.c., returning to normal levels in survivors by 2 8 days p.c.(Figure 22). The WRGPC and LISGPC inoculated animals demonstrated varying degrees of thrombocytopenia between

123

Days post-challengeVirus 0 7 14 21 28 35

WRGPC 754[50.9]

398[32.6]

306[28.4]

652[50.8]

726[32.9]

789[48.2]

LISGPC 759[48.2]

465[21.4]

392[25.3]

683[26.9]

758[32.1]

763[31.4]

LISN 732[61.2]

662[21.4]

741[21.6]

727[28.2]

789[42.9]

735[41.9]

WR 734[31.4]

375[27.6]

300[19.1]

698[31.4]

735[10.1]

728[5.2]

PBS 793[49.1]

470[28.4]

350[31.2]

579[21.2]

669[3.1]

690[4.2]

Table to Figure 22 with numbers in brackets [] showing standard error of mean.

124

Fig.22 PLATELET COUNTS IN VACCINATED ANIMALS AFTER CHALLENGE WITH LV

MEAN No. PLATELETS (1(f/L)900

800

700

600

500

400SURVIVORS

300

306392741300350

652683727698579

789763735728690

WRGPCLISGPCLISNWRPBS

754759732734793

398465662375470

726758789735669

DAYS POST INFECTION

— WRGPC LISGPC ^ LISN

WR PBS

* HORIZONTAL BARS INDICATE NORMAL RANGE OF VALUES

7 and 14 days p.c. In survivors platelet levels returned to normal by 21 days p.c. Platelet counts in LISN vaccinated animals remained within normal limits (700-820 X 10 /1) showing no evidence of a thrombocytopenia.

The majority of the recombinant LV/vaccinia virus inoculated animals (20 of 24) recovered from the thrombocytopenia. There was no significant alteration in platelet counts in the LISN animals after LV challenge.

b. NeutrophilsNormal range; 1.5-1.9 x 10*/1LV challenged control animals showed an increase in

neutrophils at 7 days p.c. (from 1.5 to 2.7 x 10^/1), with a further increase at day 14 p.c. (from 2.7 to 5.4 x 10*/1). This was the only group of animals showing this pattern (Figure 23). All other animals showed a severe neutrophilia from day 7-14 p.c.; the largest observed increase being amongst the WRGPC inoculated animals (from 0.9 to 7.1 X 10*/1) and a moderate increase in LISN inoculated animals (from 0.7 to 2.05 x 10 /1) . At all other sampling times the neutrophils were very near or below the normal range (from 1.5 to 1.9 x 10 /1) . The one exception was the PBS control group, which still had elevated neutrophil levels (2.2 x 10*/1) at 28 days p.c.

In conclusion, all groups of animals, except LISN, showed varying degrees of a neutrophilia at 14 days p.c.

125

Days post-challengeVirus 0 7 14 21 28 35

WRGPC 1.5[0.23]

0.9[0.14]

7.1[0.18]

0.96[0.26]

0.93[0.14]

0.94[0.31]

LISGPC 1.5[0.23]

1.3[0.21]

4 . 7 [0.51]

3.6[0.41]

1.3[0.14]

0.94[0.19]

LISN 1.5[0.23]

0.7[0.14]

2.05[0.24]

1.2[0.36]

1.25[0.42]

1.45[0.61]

WR 1.5[0.23]

1.2[0.12]

3.6[0.23]

1.7[0.39]

1.34[0.12]

1.44[0.12]

PBS 1.5[0.23]

2.7[0.42]

5.4[0.32]

2.5[0.27]

2.6[0.18]

2.2[0.1]

Table to Figure 23 with numbers in brackets [] showing standard error of mean.

126

Fig.23 NEUTROPHIL COUNTS IN VACCINATEDGUINEA PIGS FOLLOWING CHALLENGE WITH LV

MEAN No.NEUTROPHILS (lcf/L)

7.5SURVIVORS

6.5

5.5

4.5

3.5

2.5

0.5

7.14.72.053.6 5.4

0.940.951.45

1.4352.2

0.9 0.963.6

0.93WRGPCLISGPCLISNWRPBS

0.7 1.251.342.62.7 2.5

DAYS POST INFECTION

— WRGPC LISGPC + LISN

WR PBS

* HORIZONTAL BARS INDICATE NORMAL RANGE OF VALUES

c . LymphocytesNormal range; 6.0-7.9 x 10*/1A lymphopenia was observed in unvaccinated animals,

shown in Figure 24, by 7 days p.c. (2.4 x 10*/1), which continued until day 14 p.c. (2.4 x 10 /1) . None of the other groups of animals showed this pattern. In the WR inoculated animals there was a lymphopenia at day 7 p.c. (3.4 X 10 /1) which continued until day 14 p.c. (5.4 x 10*/1); by this time the lymphocyte levels were rising in surviving animals. By 21 days p.c. a lymphocytosis (9.8 x 10*/1) was evident, with a return to normal levels by day 35 p.c. (8.2 X 10^/1). The LISGPC inoculated animals showed a similar pattern to the WR inoculated animals.The lymphocyte levels in the WRGPC animals were close to normal, except for those observed at day 7 p.c. (2.7 x 10 /1) . No lymphopenia was observed in the LISN inoculated animals at day 7 p.c., however at days 14 and 21 p.c. a lymphocytosis was observed (8.8 x 10^/1). Similarly, the WRGPC animals showed less deviation from normal levels.

The LISN animals did show protection from a lymphopenia during the study. Animals inoculated with LISGPC and WRGPC had an early lymphopenia by day 7 p.c., returning to normal by 14 days p.c., thereafter showing varying degrees of lymphocytosis until day 28.

127

Days post-challengeVirus 0 7 14 21 28 35

WRGPC 6.8[0.93]

2.7[0.77]

7[0.39]

8.7[0.42]

8.1[0.54]

8.3[0.62]

LISGPC 6.8[0.93]

4.5[0.82]

7.2[0.71]

11.1[0.31]

10.9[0.29]

8.5[0.21]

LISN 6.8[0.93]

6.5[0.89]

8.8[0.31]

8.8[0.42]

7.8[0.83]

6.3[0.32]

WR 6.8[0.93]

3.4[0.67]

5.4[0.21]

9.8[0.42]

9.7[0.1]

8.2[0.1]

PBS 6.8[0.93]

2.4[0.32]

2.4[0.21]

6.7[0.82]

7.7[0.1]

7.2[0.1]

Table to Figure 24 with numbers in brackets [] showing standard error of mean.

128

Fig.24 LYMPHOCYTE COUNTS IN GUINEAPIGS FOLLOWING CHALLENGE WITH LV

MEAN No.LYMPHOCYTES (10/L)

SURVIVORS

35

8.7 11.18.8 9.8 6.7

8.110.97.89.77.7

8.3 8.56.3 8.2 7.2

2.74.56.53.42.4

6.86.86.86.86.8

WRGPCLISGPCLISNWRPBS

7.28.85.42.4

DAYS POST INFECTION

— WRGPC LISGPC LISN

WR PBS

‘ HORIZONTAL BARS INDICATE NORMAL RANGE OF VALUES

d. LeukocytesNormal range: 7.8-9.4 x 10*/1In unvaccinated animals there was a leukopenia at

day 7 p.c. (5.4 X 10*/1) with a return to normal levels at day 14 p.c. (Figure 25). After day 14 p.c. leukophilia was observed. There was a leukopenia in the WR inoculated animals at day 7 p.c. (4.8 x 10 /1) with reversal to leukophilia by day 14 p.c. (10.15 x 10*/1) and this leukophilia persisted to day 35 p.c. A maximum of 12.2 x 10*/1 leukocytes was attained at 21 days p.c.

With both the LISGPC and the WRGPC inoculated animals a leukopenia was observed at day 7 p.c.; the WRGPC animals showing the most pronounced leukopenia of all the animal groups (3.9 x 10 /1) . There was a reversal in both the LISGPC and WRGPC inoculated animals at day 14 p.c to leukophilia (11.7 x 10^/1 and 14.2 x 10^/1 respectively). Although the WRGPC inoculated animals showed the most severe leukopenia and severe leukophilia, they had reverted to near the normal range by day 21 p.c. and thereafter stayed near normal levels. Leukophilia persisted in the LISGPC inoculated group until day 28 p.c. (11.28 X 10 /1) and was almost within the normal range by 35 days p.c. No leukopenia was observed in the LISN inoculated animals, although there was leukophilia at 14 and 21 days p.c. (11.7 and 10.57 x 10*/1 respectively).

Animals displayed an early leukopenia, followed by leukophilia at day 14 p.c. All groups of animals showed a

129

Days post-challengeVirus 0 7 14 21 28 35

WRGPC 8.61[0.64]

3.9[1.42]

14.2[2.92]

9.67[2.47]

9.43[2.12]

9.51[2.5]

LISGPC 8.61[0.64]

6.2[2.41]

13.1[1.41]

15.12[3.46]

11.28[3.11]

9.54[1.69]

LISN 8.61[0.64]

8.2[1.91]

11.7[1.33]

10.57[1.45]

8.82[1.83]

8.14[2.2]

WR 8.61[0.64]

4.8[0.93]

10.15[2.24]

12.2[0.54]

11.47[0.45]

10.05[0.56]

PBS 8.61[0.64]

5.4[1.81]

8.62[1.97]

11.8[1.91]

11.1[1.5]

9.9[1.5]

Table to Figure 25 with numbers in brackets [] showing standard error of mean.

130

Fig.25 LEUKOCYTE COUNTS IN VACCINATEDGUINEA PIGS FOLLOWING CHALLENGE WITH LV

LEUKOCYTES (10/L)

SURVIVORS

9.519.548.1410.059.9

14.213.111.7

10.158.62

8.618.618.618.618.61

3.96.28.24.85.4

9.6715.1210.5712.211.8

9.4311.288.8211.47

WRGPCLISGPCLISNWRPBS

DAYS POST INFECTION

— WRGPC LISGPC LISN

WR PBS

* HORIZONTAL BARS INDICATE NORMAL RANGE OF VALUES

return to normal leukocyte levels at different times p.c., except WR inoculated animals where the leukocyte levels were still outside the normal range at 35 days p.c.

8. Biochemistry

In LF human patients a serum aspartate aminotransferase (AST) level greater than 150 IU/1 is associated with a case fatality of 50% (Johnson et al., 1987). There is also good correlation between elevated AST levels and an increased risk of death. For these reasons the serum AST levels were studied in the guinea pigs. Serum alanine aminotransferase (ALT) levels were also measured.

a. ASTNormal range: 60-85 IU/1After LV challenge there was a similar pattern of

serum AST levels in the unvaccinated and WR inoculated animals (Figure 26). At day 7 p.c. the AST levels were extremely high in both groups (235 IU/1 in PBS animals and 210 IU/1 in WR animals). By 14 days p.c. the levels remained very high (189 and 200 IU/1 respectively). A return to near normal levels was observed at 21 days p.c in surviving animals in these two groups, and the AST levels remained normal until the end of the experiment.

131

Days post-challengeVirus 0 7 14 21 28 35

WRGPC 75 155 110 85 70 78[15.4] [19.4] [10.8] [12.6] [14.9] [15.2]

LISGPC 75 180 125 86 73 73[15.4] [20] [16.3] [14.2] [12.8] [12.4]

LISN 75 160 130 89 75 69[15.4] [23] [16.3] [12.2] [11.1] [3.2]

WR 75 210 200 95 80 73[15.4] [28.9] [32.8] [12.4] [9.8] [4.9]

PBS 75 235 189 80 85 79[15.4] [20] [31.4] [12.4] [5.2] [4.2]

Table to Figure 26 with numbers in brackets [] showing standard error of mean.

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Fig.26 SERUM AST LEVELS IN VACCINATEDGUINEA PIGS FOLLOWING CHALLENGE WITH LV

MEAN AST (lU/L)250

SURVIVORS

200

150

100

50

110125130200189

155180160210235

WRGPCLISGPCLISNWRPBS

73 73

DAYS POST INFECTION

— WRGPC LISGPC LISN

WR -X— PB3

* HORIZONTAL BARS INDICATE NORMAL RANGE OF VALUES

In all groups of recombinant vaccinia virus inoculated guinea pigs (WRGPC, LISGPC and LISN) there were elevated AST levels at day 7 p.c.; the highest level being 180 IU/1 in the LISGPC animals. By 14 days p.c. all levels were reduced to between 110 and 130 IU/1 and had returned to normal levels by 21 days p.c.

b. ALTNormal range: 40-60 IU/1All groups of animals showed similar patterns in ALT

levels after LV challenge (Figure 27). The levels were highest in the PBS. and WR inoculated animals, with levels of 120 and 118 IU/1 respectively at 7 days p.c. Levels in the recombinant vaccinia virus inoculated animals were not as high (from 98 to 110 IU/1), although the values were still double the normal levels. By 14 days p.c. there was a reduction in ALT levels in all groups of animals (the highest was the WR animals at 110 IU/1), although these were still outside the normal range. There was a return to near the normal range by 21 days p.c.(the highest level in the WR animals at 75 IU/1).

9. Post challenge anti-LV antibody levelsAt 7 days after LV challenge, serum anti-LV levels

were undetectable in most animals (Figure 28). The only group of animals that began to show an anti-LV IgG titre were the LISN animals, in which the titre was greater

133

Days post-challengeVirus 0 7 14 21 28 35

WRGPC 49.6[9.4]

110[7.1]

95[8.6]

56[8.6]

60[8.7]

49[7.3]

LISGPC 49.6[9.4]

100[6.2]

90[5.9]

63[3.1]

57[8.4]

60[7.1]

LISN 49.6[9.4]

98[4.1]

84[6.2]

67[7.1]

59[8.2]

47[9.1]

WR 49.6[9.4]

118[3.6]

110[10.2]

75[6.1]

67[0.12]

56[0.12]

PBS 49.6[9.4]

120[8.1]

109[10.4]

40[0.1]

55[0.1]

59[0.3]

Table to Figure 27 with numbers in brackets [] showing standard error of mean.

134

Fig. 27 SERUM ALT LEVELS IN VACCINATEDGUINEA PIGS FOLLOWING CHALLENGE WITH LV

MEAN ALT (lU/L)160

140 SURVIVORS

120

100

80

11010098118120

49.649.649.649.649.6

WRGPCLISGPCLISNWRPBS

6384110109

56

DAYS POST INFECTION

— WRGPC LISGPC LISN

WR PBS

* HORIZONTAL BARS INDICATE NORMAL RANGE OF VALUES

than l/lOO. By 14 days p.c. all groups of animals had titres greater than 1/1,000 and by 21 days p.c. all had serum anti-LV titres greater than 1/1,000,000.

There were no detectable anti-LV IgG titres prior to challenge: the detectable levels in the LISN animals at 7 days p.c. could be because the antibody used in the ELISA is purified LV and N is the most abundant protein in the virion. There is no readily available explanation for the high IgG levels at 14 and 21 days p.c. in all the groups of animals. To verify the results immunofluorescence tests were carried out on the serum and these tests confirmed the very high ELISA results of 1/1,000,000. No neutralising antibody was detected in any guinea pig serum.

10. Virus levelsLV was detected in the serum of all groups of

animals between 4 and 10 days p.c. (Table 14). The highest level was in the PBS virus control group where a titre of 10 to 10 p.f.u./ml was detected. A lower titre of 10 to 10 p.f.u./ml was present in the serum of WR animals. All the recombinant vaccinia virus groups had serum titres of 10 to 10 p.f.u./ml. This demonstrated that although LV virus titres were reduced in vaccinated guinea pigs, the vaccination did not completely prevent virus replication.

135

Fig.28 SERUM ANTI-LV ANTIBODY TITRES IN GUINEA PIGS FOLLOWING CHALLENGE WITH LV

Anti-LV ELISA IgG antibody

1 .OOOE-i-07

1 .OOOE+06

1 .OOOE+05

1 .OOOE+04SURVIVORS

1 .OOOE+03

1 .OOOE+020 7 14DAYS POST INFECTION

21

— WRGPC

-B- WR

LISGPC LISN

PBS

136

O O 'f M H ■H

M -H -d JJ iH 'S' d -H H Q) f! I a OiO

O d D A4 -H d fl r-0) (D I J OiO

I—1-H ■H -H-H

-H0) -P

4-> -H nj 4->01

0) rHCO 0)

■H -d > 0) 733 -0 g o

VD

4-14-1 nn

0) H 4-1 (U CN0) H(D H

<U I> U H•H i-sX «1 0) >1 M fS >i73

5-10) H5-1 (NQJ r4

54 H Q) H > O 1) HO 00

V 0) A3 d) d) ti -H rt 0) A h -H-H ■H

■H

4-)fd0TlIWo&T5Ürd0B

1

137

c. DISCUSSIONGuinea pigs immunized with the recombinant vaccinia

viruses and the wild-type WR vaccinia virus displayed no adverse effects to the immunization. Areas covered by the lesions and scabs produced by the virus were generally smaller with the recombinant vaccinia viruses than the wild-type virus, even though the same infectivity titres were used for inoculation. After the second scarification, that was given to half the animals, even smaller lesions and scabs occurred, in comparison with the first scarification. By 4 weeks post-immunization all guinea pigs inoculated with either wild-type vaccinia virus or the recombinant vaccinia viruses, had produced antibodies to vaccinia virus. There was a slight increase in the antibody response at 8 weeks post-immunization. No difference in the antibody levels was detected in the animals receiving either one or two scarifications. No anti-LV antibodies were detected in any guinea pig prior to LV challenge.

Two guinea pigs survived challenge with LV in each of the two control groups, PBS and WR vaccinia virus. Increasing the dose of the LV challenge would not have increased the mortality (Jahrling et al., 1982) and in these surviving animals more severe effects of disease were observed than in any of the animals in the recombinant vaccinia virus groups (Table 14). All three LV recombinant vaccinia virus constructs, WRGPC, LISGPC and LISN, tested in this experiment afforded some degree

138

of protection to the guinea pigs. Surprisingly, the LISGPC (no deaths) gave better protection than the WRGPC (25% mortality), because recombinants made with the WR strain of vaccinia virus often give the best protection (Hruby, 1990). Protection with the LISN construct was good (12.5% mortality). Combining the mortality data from the control groups (PBS and WR) and the recombinant vaccinia virus groups (WRGPC, LISGPC and LISN), shows that 4 of 16 (25%) guinea pigs survived challenge in the control group, whereas and 21 of 24 (87.5%) survived challenge in the vaccinated groups.

The results from the guinea pigs given two scarifications are not presented, because there was no difference in the data from those animals receiving a single immunization. These results were reproducible in two separate experiments.

During the acute phase of LV disease, between 0-14 days p.c., the weight of most of the guinea pigs was static. The exception was the PBS group, where an increase in weight occurred between days 6 and 8 p.c., followed by a sudden weight loss (Figure 20). After 10 days p.c. most animals showed some weight loss, although with most of the recombinant vaccinia virus animals the decrease in weight was minimal. In the LISN animals, a rapid increase in weight was observed after 10 days p.c. and the WRGPC animals were also mainly protected from anorexia.

139

Increased temperature was observed in all the groups of animals at some time during the first 10 days p.c.(Figure 21). The PBS and WR control groups showed an increase in temperature to greater than 40°C followed by a rapid temperature fall to below 38°C. This shock syndrome is associated with fatalities in human Lassa fever patients. Only after 16 days p.c. was there a gradual return to normal temperatures, with these guinea pigs. With the LISGPC and WRGPC animals fever occurred, although temperatures did not fall below normal values. There was the least variation in temperatures in the LISN guinea pigs.

The haematology is compared and discussed throughout the results section and summarised in Table 14. Immunization with the LISGPC and WRGPC viruses did not prevent thrombocytopenia and neutrophilia, although immunization with the LISN did. Lymphopenia was moderated with the recombinant vaccinia virus inoculated animals and leukopenia was not severe in either the control or the recombinant vaccinia virus inoculated animals.

Normal AST levels were 75 IU/1 in these guinea pigs and by 7 days p.c. this had risen to over 200 IU/1 in the two control groups (PBS and WR). With the recombinant vaccinia viruses, although the AST levels were increased at 7 days p.c., they were falling to close to normal levels by 14 days p.c. (Figure 26). At 14 days p.c. the AST levels in the control groups were over 180 IU/1.Serum AST levels of above 150 IU/1 have been correlated

140

with increased risk of death in human Lassa fever patients and this association seems to be applicable to this guinea pig model. High AST levels were similarly, though less dramatically affected and moderated in the vaccinated guinea pigs.

Anti-Lassa IgG antibody ELISA titres were observed in all surviving animals at day 14 p.c. (Figure 28), with levels rising to >1 x 10 at 21 days p.c. It has been previously documented that guinea pigs surviving LV challenge develop high antibody titres to LV post challenge (Jahrling et al., 1982). Fatalities from LV infections in humans have been correlated to high virus titres, however no relationship has been established with the anti-LV antibody levels (Johnson et al., 1987) .

141

CHAPTER 4

CONCLUSION

REFERENCES

CONCLUSIONUntil 10 years ago, successful vaccines were

frequently prepared from inactivated or attenuated pathogens. Now, with increased awareness of health and safety and the soaring costs of litigation, alternatives to these processes for vaccine manufacture are being sought. Potential recombinant vaccines, where only a small part of the pathogens genome is used, should reduce the risks of possible side-effects to the recipients.With LV, conventional inactivation methods using gamma- irradiation or formaldehyde treatment, have resulted in products that failed to provide protection against LV challenge (Morrison et al., 1989). Given a pathogen with the hazardous nature of LV, where little is known about the immunogenicity of the virus structural proteins and studies are restricted because of the containment required for handling the virus; a recombinant vaccine may be very advantageous.

There are three proteins that are major components of the LV structure, however which protein is the best immunogen is not known. Previous researchers (Clegg and Lloyd, 1987; Auperin et al., 1988; Morrison et al., 1989) have used recombinant vaccinia virus constructs made with either the LV N protein or the precursor glycoprotein (GPC). It would have been of interest to compare the protection provided by a recombinant vaccinia virus made with either the LV G1 or G2 glycoproteins, as there is highest conservation between the amino acid sequences of

142

G2 arenavirus proteins and G1 is closer to the virion surface (Burns and Buchmeier, 1991). However it was not possible to make these separate vaccinia constructs (Dr. Chris Clegg, personal communication).

Although there have been only three previous protection studies with recombinant vaccinia viruses containing LV gene inserts using guinea pigs, it is impossible to compare these studies directly because different conditions were used for each experiment. These variables include the LV strain for challenge, the time interval between vaccination and challenge, and the strain of vaccinia virus for making the construct. Very few parameters of the disease were assessed in any of the experiments, although partial protection was obtained with each construct. Morrison et al. (1989), did compare protection provided by recombinant vaccinia viruses containing inserts of the N or GPC gene under the same experimental conditions. These researchers obtained better protection with N (94% survivors) compared with GPC (79% survivors).

The original intention of this current study was to compare protection provided by the LV GPC gene vectored by two different vaccinia virus strains (Lister and WR). When a recombinant vaccinia virus containing the LIS N gene became available, this was also included in the protection studies. All constructs gave some degree of protection. It was found that the LISGPC gave the best protection (100%), the LISN the next (87.5%) and then

143

WRGPC (75%). This compares with a 25% survival rate in the control groups. In this study, to test the long-term efficacy of any immunization, a longer time interval was allowed to elapse between immunization and LV challenge, 56 days post-immunization compared with 21 days (Morrison et al., 1989 and Auperin et al., 1988) and 28 days (Clegg and Lloyd, 1987) in previous reports. This suggests that long-term immunity is induced by the recombinant vaccinia viruses. There was no difference in survival or disease in any animal receiving two immunizations at an interval of four weeks, so short term booster immunizations are unnecessary.

A detailed comparison of the disease profiles of Hartley Dunkin guinea pigs after LV challenge in vaccinated and unvaccinated animals has not previously been reported. When protection from the effects of disease in the recombinant vaccinia virus immunized animals was compared, the LISN gave the best protection. All three recombinant vaccinia viruses, WRGPC, LISGPC and LISN, moderated the severity of the disease in comparison with the control animals. The serum AST levels, reported to be a critical factor in fatalities from LV, were lower in comparison with the control groups and rapidly returned to normal levels.

All LV challenge studies to date with recombinant vaccinia virus immunized animals have used the intraperitoneal route for challenge. The spread of LV has been demonstrated to occur by the aerosol route in the

144

laboratory and infectivity has been shown to occur in primates and guinea pigs by this route (Peters et al., 1987). A study into the feasibilty of protection from this route of challenge would be worthwhile. The main contributing factor in causing death in LV infections has yet to be established. As different internal organs are damaged by LV infections induced by aerosolised virus (Peters et al., 1987), it is possible that fatalities occur by disfunction of different physiological mechanisms.

The Lister and NYBH strains of vaccinia virus were both attenuated stains of virus approved by WHO for human vaccination in smallpox protection. For this reason, researchers have used these two strains to make the LV recombinant vaccinia virus constructs. Vaccinia virus WR strain is more virulent and its recombinants have often induced better protection than recombinants made with the more attenuated strains (Morgan et al., 1988; lacono- Connors et al., 1991). In this study, a comparison was made of recombinants prepared with the Lister strain and the WR strain of vaccinia virus. The foreign gene used in the recombinants was the LV precursor glycoprotein gene and the insert was made into the TK gene of the vaccinia virus genome. Better protection was obtained against LV challenge with the LISGPC recombinant (100%) than with the WRGPC recombinant (75%). There is no obvious explanation why this should happen. Both constructs showed similar expression levels in vitro, with the WRGPC

145

displaying stronger immunofluorescence levels. A similar moderation of LF disease parameters occurred with both constructs.

Increased safety regulations have raised doubts that these attenuated vaccinia virus strains, Lister and NYBH, would be allowed for human immunization in the future. With the sequencing of the Copenhagen strain of vaccinia virus and the identification of virulence genes in the vaccinia genome, new attenuated strains are being produced. Both NYVAC and LC16m8 strain recombinants have induced protection against pathogens in animals, and future recombinants should be made with similarly attenuated strains. Alternatively the avipox virus,ALVAC, would be an interesting vector.

Therefore, in conclusion, this thesis has demonstrated that recombinant vaccinia virus expressing LV genes can protect guinea pigs from LV challenge. Clearly, these are only preliminary experiments and further studies are necessary to exploit this system to investigate a potential LF vaccine, induction of protective immunity by LV antigens and the pathogenesis of LV.

146

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