Future directions

Conclusions about LN FNA procedure

LN FNA feasibility pilot study (n=5)

LN FNA HPV vaccination study (n=15)

HPV-specific assay development

Conclusions about clinical research

CONCLUSIONS & OUTLOOK

9 weeks with SURP

First LN FNA

ACKNOWLEDGEMENTS

STUDY PROGRESS OBJECTIVES

INTRODUCTION

LOGISTICS

METHODS & RESULTS

Evaluating lymph node fine needle aspiration (LN FNA) for characterization of immune response to vaccination

Cambray Smith1,4, Austin Varni1, Jody Carter2, Denise Galloway2, Janine Maenza1,3, Julie Czartoski1,3, Antje Heit1 and Julie McElrath1,3

1Fred Hutchinson Cancer Research Center, Vaccine and Infectious Disease Division, Seattle, WA 2Fred Hutchinson Cancer Research Center, Human Biology Division, Seattle, WA

3Seattle Vaccine Trial Unit, Seattle, WA 4North Carolina State University, Raleigh, NC

Lymph node (LN)

Primary follicle

Germinal Center

(GC)

Week -12 to 0

Week 0

Week 3

Week 8 Week 11

Month 6

Screen (HPV -16 & -18)

Vax 1 LN

FNA Vax 2

LN FNA

Vax 3

Deltoid vax w/ axillary FNA (n=10)

Quad vax w/ inguinal FNA (n=5)

6

15

73

0 20 40 60 80

Indeter for 1+

Positive for 1+

Negative for all types

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21

-16

-18

-11

-33

Funding: SURP is supported in parts by the Cancer Center Support Grant (CCSG) CURE Supplement: NCI 3 P30 CA0157043P30CA015704-42S4 and the Fred Hutch Internship Program. Project funding comes from CTU (clinic) and HIV Vaccine Trials Network (HVTN) grants.

Acknowledgements: Thank you to Julie McElrath, the Tfh group (Antje, Austin, Saba, and Tayler), the SVTU clinic, the Galloway lab, the Eastlake summer students/immunology class members, Andrea Musa, Rachael Nelson, Niki Robinson, the SURP coordinators, and all of the study volunteers.

Traditionally, vaccine efficacy in humans is assessed by sampling peripheral blood.

After vaccination, germinal centers (GC) form in follicles of draining lymph nodes (LN). In GC, B cell differentiation, guided by T follicular helper (Tfh) cells, takes place. The GC reaction process creates high-affinity antibodies of vaccine-specific B cells which results in protective and long-lasting immunity.

Directly sampling LN may provide access to early vaccine-induced immune responses not seen in peripheral blood, which could be predictive of vaccine efficacy.

Lymph node fine needle aspiration (LN FNA) could be an important clinical procedure for characterizing functional immune response to novel vaccines, such as HIV vaccines. The procedure is performed with a needle inserted under ultrasound, and multiple “passes” may be taken to try to ensure collection of GC cells.

To incorporate LN FNA in clinical research, complex coordination is key and requires substantial management, logistical planning, and coordination. The LN FNA procedure was tested in a small feasibility pilot study (n=5) followed by testing in a HPV vaccine study (n=15).

1. Understand the complex coordination and collaboration required for beginning a new clinical study.

2. Explain progress and preliminary results of a multi-phase clinical study which aims to

a) Evaluate the feasibility of lymph node fine needle aspiration (LN FNA) in a research setting, and

b) Determine if the procedure can provide additional insight in characterizing functional immune response to human vaccination by monitoring response to HPV vaccination.

Is the LN FNA procedure feasible, safe, and tolerable in a research setting?

Can LN FNA be used to access vaccine-specific GC B and T cells?

Dark zone

T cell zone

Mantle zone

20X

Light zone

Clinical research requires collaboration and cooperation between basic and clinical researchers, clinicians, hospitals, administrators, regulatory teams, community-builders, and research volunteers.

New clinical studies take months/years to develop and implement.

Working with human volunteers requires extensive time and effort.

Participants

Volunteer for studies; make

a commitment to research

Scientists

Ask questions, set visions, and steer

direction of studies

Ensure ethical

studies and keep records

Regulatory

Partner with participants,

often scientists, procure samples

Clinicians

Admin

Coordinate between groups

Lab Techs

Process samples and run lab tests

Figure 3. Foundations of a clinical study Figure 4. LN FNA Collaboration

Processing of samples Analysis of B and Tfh

cells

McElrath Lab, Fred Hutch

HPV antibody screening

Galloway Lab, Fred Hutch

LN FNA procedures

Harborview Medical Center

Seattle Vaccine Trials Unit

Clinical procedures Lab analyses

Figure 2. LN FNA probing GC Figure 1. GCs in human tonsil tissue

Jul

Tim

elin

e o

f st

ud

y d

eve

lop

me

nt,

Ju

n 2

01

6-p

rese

nt

Study population:

Prior Seattle Vaccine Trials Unit participants

Healthy, young1

Methods:

Clinical: 1 LN FNA procedure

Laboratory: flow cytometry (BD Fortessa X50 and Aria cell sorting)

Results:

Participants did not report any adverse effects; procedure was well tolerated

McElrath lab preparation for HPV phase includes assay refinement (X50 panels, Aria cell sorting).

Screen # HPV-16 HPV-18 Eligible?

1 + + No1

2 - - Yes

3 - - Yes

1Values that are positive for 1+ HPV types are excluded from the indeter. category in the HPV seropositivity graph.

Key: Positive (≥1000 MFI) Indet (500-1000 MFI)

Table 3. Coinfections of HPV types 111, 16, 18, and 33

94 serum samples from the Seattle Assay Control cohort were screened for HPV types 11, 16, 18, and 33

Samples being used to develop HPV-specific flow panels for GC B and GC Tfh cells

Study population:

Healthy , ages 18-21

No Hx of HPV vaccination

Seronegativity for HPV-16 and -18

Methods:

Serum screened with HPV antibodies (IgG) binding test (Galloway Lab)

Randomized into two groups (Fig. 5) and vaccinated with Gardasil-9 (Tab. 2)

Lab assays: X50 flow phenotyping, Aria cell sorting, HPV-specific GC cell genetic analysis

Preliminary screening results:

1Although volunteer 1 was ineligible for HPV study, donor chose to participate in LN FNA pilot cohort as 5th LN FNA donor.

1HPV 16 & 18 are the only

strains for inclusion/

exclusion for HPV study,

but 11 & 33 also tested in

standard screening.

Figure 5. Vax/LN FNA location1

1If draining LN cannot be accessed,

radiologist will follow anatomic hierarchy as

laid out in protocol.

Tayler Fluharty

Table 2. HPV study schedule

Table 3. HPV screening

Figure 7.

1

Positive ≥1000 MFI Indet 500-1000 MFI Recruit/enrollment

Vax & specimen collection

LN FNA donor

Site Cell

number B Cells T Cells

1 Inguinal 0.8M 20% 55%

2 Axillary 0.9M 15% 47%

31 Inguinal 0.56M 1.6% 17%

42 Inguinal 0.1M 0.3% 0.3%

5 Inguinal 5M 39% 8.5%

1Likely LN was missed. 2Likely no suction was applied.

Table 1. LN FNA from non-vaccinated donor

Figure 6. HPV seropositivity

Outreach

Build relationships

with communities

Funding

Grant writing and

partnership building

1Goal was “as young as possible” (>18). Ages ranged from 21-42 y.o. 2Males, females, and transgender individuals.

2

In our study, up to 4 LN FNA passes may be taken.

How do we prepare an HIV lab to isolate HPV-specific B and T cells?

LN FNA as a research procedure in humans is well-tolerated.

LN cells participating in GC reactions can be accessed, sampled and studied for protective B cell responses.

Moving into HPV vaccination stage – the study requires up to 9 months to complete, so finding volunteers in a confined age range (18-21 y.o.) may prove challenging.

Ultimately, the LN FNA procedure will be incorporated into HIV candidate vaccine evaluation to inform longevity and specificity of HIV vaccine immune responses.

First donor screen

First donor screen

Jun 2016: Discussion of LN FNA project

Aug

2016

2017

Mucosal protocol modified - IRB

Clinical approval for pilot (Harborview)

Jun

LN FNA pilot recruitment

Clinical approval for HPV study (Harborview)

Final pilot

LN FNA

HPV recruitment

Efforts to establish assay procedures

began in Fall 2016 and continue to present.

Collaboration with Galloway

Lab (HPV) confirmed

May Apr Mar Feb Jan

Jun Jul Aug Sep Oct Nov Dec