Challenges of Integrating Mucosal Immune Assays into HIV Vaccine Trials Omu Anzala, MBChB, PhD KAVI University of Nairobi
Outline Why mucosal immunology Experience from Kenya Challenges The way forward
Background Sexual transmission of HIV represents the dominant mode of transmission globally Often a single virus transmits across the genital mucosa and infects a target cell within the genital lamina propria An effective vaccine should elicit immune response at mucosal sites as these are sites of viral entry Understanding immune response at the mucosal sites has now became critical for HIV vaccine development
HIV vaccine trials A number of vaccine trials have attempted to assays mucosal responses following vaccine administration Results have varied depending on: The type of vaccine Route of administration Sites sampled 4
Approaches Developing KAVI as a Centre of Excellence in Mucosal Immunology With Funding from IAVI, KAVI has embarked on number of studies on Mucosal immunology: - Genital study initiated in 2009 to develop mucosal sampling methods and assays to assess mucosal immune responses in GU tract - Invasive mucosal sampling initiated in 2011 to pilot HIL methodologies to assess cellular responses in GALT (colon) Haas et al Nature 2010 - Application into clinical trials. initiated in 2011- mucosal sampling in vaccine trials
Mucosal immunology studies at KAVI Studies on mucosal immunology: - Protocol J initiated in 2009 - Protocol M initiated in 2011 - Protocol I initiated in 2011 6
Mucosal specimen collection in Africa; a pilot study to find out the feasibility of mucosal sampling methods for use in future HIV vaccine trials. (Protocol J)
Objectives Primary objectives: To assess the feasibility of mucosal sampling methods in an African setting To assess the immune response in ESN volunteers and to compare it to a group of volunteers with lower risk of exposure to HIV infection and a group of HIV sero-positive volunteers 8
Volunteers enrolled A study was designed to recruit volunteers in different groups: - High risk sero-negative (HRN) volunteers - Low risk sero-negative (LRN) volunteers - HIV infected volunteers 5 volunteers in each group sampled at monthly intervals for 4 visits 9
Genital Sampling Sampling consisted of: - Males - serum, semen, rectal cytobrush/sponge - Females serum, cervical cytobrush/sponge, rectal cytobrush/sponge 14/15(93%) provided rectal samples at study entry Samples evaluated for cellular and antibody responses. f Lessons learned at KAVI were used to modify the protocol and repeated at a second Kenyan research centre (KEMRI Kilifi), where 15 more volunteers were enrolled
Rectal sampling was poorly accepted in low risk volunteers at KAVI Due to the Cytobrush? 5 N umber of Vo olunteers Giv ving Sample es 4 3 2 1 ESN, n=5 Low Risk, n=5 HIV+, n=5 0 M0 M1 M2 M3 Time Points
Anti HIV-1-gp140 antibodies can be detected in genital fluids IgG IgA Male Female Male Female (n=24) (n=6) (n=24) (n=6) Cervical Sponge -- 2/6 -- 0/6 Seminal supernatant 8/18 -- 0/18 -- Rectal Sponge 8/19 2/6 4/19 1/6 Serum 10/24 2/6 10/24 3/6 - No antibodies were detected in any low risk seronegative volunteer samples - IgG and/or IgA was detected in serum from some high risk seronegative volunteers- who remained PCR negative through the 4-month study
Is it possible to perform genital sampling? Volunteers accept repeated sampling of the genital mucosa- dependent on the type of sampling and the group. Low risk seronegatives are least likely to provide samples. Cytobrushes did yield cell numbers that t could be phenotyped, but were too few in number for functional assays: - rectal mean=120,000 cells - cervical mean= 100,000000 cells Merocel sponge collection was sufficient to capture luminal HIV-1- specific antibodies. HIV-1-specific IgG, IgA or both were observed from cervical and rectal eluates and in semen e supernatants ts 13
A prospective observational study to detect cellular immune function in gastro-intestinal mucosal tissues and peripheral blood (Protocol I)
Objectives To measure cellular immune function in lower gastro-intestinal mucosal tissues obtained via biopsies of the colon To compare cellular immune responses in the mucosa and peripheral blood of HIV sero negative volunteers with that of HIV-infected individuals Over the last 4 years the IAVI Human Immunology Laboratory (HIL) has optimised i methodologies for assessing anti-hiv T cell responses in gastric tissue, that may be standardised and quality controlled for use in Vaccine trials. (Kaltsidis et al. J Immuno Methods 2011) Utilising the XXXX LSRII in place at the University of Nairobi, these methods have been transferred into KAVI 15
Methods: Isolation of mucosal mononuclear cells (MMC) from gut Upto 10 pinch biopsies using jumbo forceps collected into MR15 media (RPMI, 15%FCS, with antibiotics/antifungals) (rectum, colon and/or duodenum (HIL) Stimulate with antigen and perform flow cytometry (Elispot) Centrifuge, remove supernatant. t Add collagenase containing digestion media and agitate t Centrifuge free cells and resuspend in MR15. Repeat Place remaining tissue fragments in digestion media and agitate Pool & wash cells, viable count and overnight rest Based on Shacklett protocol (no percoll gradient separation, overnight rested in different media/antibiotics)
KAVI generate viable cells from colonic biopsies KAVI get 10-12 biopsies from each volunteer The average yield is approx. 1.11 x10 6/ biopsy. Similar to IAVI- HIL and others (i.e. BL Shacklett)
Cellular markers being assessed at IAVI-HIL and KAVI HIL Mucosal Panel Viability Dye (ViViD) CD3 QD656 CD4 QD606 CD8 Pacific Orange CD19 Pacific Blue (Dump) CD14 Pacific Blue (Dump) CD107a PECy5 IL 2 APC TNF A700 IFN PECy7 KAVI Mucosal Panel Viability Dye (Aqua Blue) CD3 APC Cy7 CD4 PETR CD8 Pacific Blue CD19 AmCyan (Dump) CD107a PECy5 IL 2 PE TNF FITC IFN APC CD45RO A700 CD27 PECy7 Panel optimized for LSRII available to KAVI from that used at IAVI-HIL Method transferred from IAVI- HIL and qualitative ti equivalence demonstrated using a blinded panel of PBMC and stimuli
KAVI and HIL generate equivalent data in HIV negative MMCs 0.8 KAVI, IAVI- HIL 0.6 % Expression 0.4 0.2 0.0 CD107+ CD107+ IFNg+ IFNg+ IL-2+ IL-2+ TNFa TNF-A+ Cytokine CD8 T cell responses to SEB stimulation CD8 T-cell responses to SEB stimulation HIV seronegative volunteers from London (HIL) or Nairobi (KAVI) Using respective panels See Kaltisidis et al 2011 for details
KAVI generates robust data in fresh MMC MOCK SEB 250K 250K 250K 250K 200K 200K 200K 200K SSC-A 150K 100K IFN-G+ 0.27 SSC-A 150K 100K TNF-A+ 0.12 SSC-A 150K 100K SSC-A 150K 100K TNF-A+ 10.7 CD4+ 50K 50K 50K 50K 0 0 10 2 10 3 10 4 10 5 <APC-A>: IFN 0 0 10 3 10 4 10 5 <FITC-A>: TNF 0 IFN-G+ 7.43 0 10 2 10 3 10 4 10 5 <APC-A>: IFN 0 0 10 3 10 4 10 5 <FITC-A>: TNF 250K 250K 250K 250K SSC-A S 200K 150K 100K 50K 0 IFN-G+ 0.12 0 10 2 10 3 10 4 10 5 <APC-A>: IFN SSC-A S 200K 150K 100K 50K 0 TNF-A+ 0.34 0 10 3 10 4 10 5 <FITC-A>: TNF SSC-A S 200K 150K 100K 50K 0 IFN-G+ 18.9 0 10 2 10 3 10 4 10 5 <APC-A>: IFN SS SC-A 200K 150K 100K 50K 0 TNF-A+ 10.4 0 10 3 10 4 10 5 <FITC-A>: TNF IFN- TNF- IFN- TNF- CD D8+ CD3 gated population
Ongoing g Vaccine Studies (2011-2012) B002? Clinical i l trial to be conducted d in partnership with at Glaxo Smith Kline (GSK) Phase 1 study Test different combinations of Ad35- GRIN and F4co/AS01 Completed p vaccinations:( 35 enrolled at KAVI) B003? Clinical i l trial to be conducted d in partnership with at Beth Israel Deaconess Medical Center (BIDMC) Test different combinations of recombinant adenovirus Ad26 vector HIV vaccine and recombinant Ad35 Fully enrolled:40 at KAVI Multi-centre- Boston, Kigali, S/Africa (3 sites)
Applications to HIV Vaccine Clinical Trials Based on the optimization of sampling methods and recruitment from the initial pilot studies, sampling with rectal and cervical sponges is currently ongoing in two Phase 1 IAVI-sponsored vaccine trials at KAVI. Saliva and semen samples are also being collected Volunteers enrolling into the vaccine trial are separately consented to provide mucosal samples Volunteer acceptability of mucosal sampling methods is being assessed Samples will be collected at pre-vaccination, and two post-vaccination time points and assayed for antibody responses 22
Challenges in assessing Cellular responses in GI tract Invasive sampling techniques requires GI facilities, collaborations with GI surgeons, and trained staff Flow cytometry requires trained personnel Flow panels needed to be optimized and qualified in PBMC Responses in Kenya might be different Infection, inflammation, nutritional status
Summary Repeated mucosal sampling can be performed Rectal sampling is the least well tolerated of the methods assessed Low risk seronegative volunteers are the least likely to give samples Extensive community enrollment, and counseling, appeared to increase mucosal sampling acceptance Detection of IgG and IgA anti-hiv gp 140 antibodies in mucosal secretions collected with Merocel sponge suggests this method could be applied to clinical trials Small number of MMCs isolated from cytobrush samples indicates the need to assess alternative methods for functional assays (invasive procedures) 24
What next? Application to clinical trials Based on the optimization of sampling methods, and recruitment from the initial pilot studies sampling with a rectal and cervical Merocel sponge has been applied into two Phase 1 IAVI sponsored vaccine trials ongoing at KAVI; In addition oral saliva and semen (male) samples are also collected These trials occur at two sites and volunteers enrolling into the vaccine are separately consented to provide mucosal samples To-date over 70 volunteers have agreed to give at least 2 mucosal sample types Samples are collected at pre-vaccination, and two postvaccination time points 25
Acknowledgements ( IAVI, Kilifi and KAVI Staff) & Volunteers taking part in the studies
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