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Montefiori Progress Report Abstracts continued

 
Submitted December 21, 2010 (Interim Report)

The Comprehensive Antibody Vaccine Immune Monitoring Consortium (CA‐VIMC) utilizes valid laboratory criteria and Good Clinical Laboratory Practices (GCLP) to assess systemic and mucosal antibody responses in preclinical and clinical phases of vaccine testing. We also seek new scientific insights, including correlates of protective immunity, that will help bridge the gap between preclinical vaccine discovery and human clinical trials. Highly standardized and validated assays are used to assess neutralizing antibodies in a GCLP‐compliant manner. Neutralizing antibodies are assessed in multiple cell types (e.g., TZM‐bl, A3R5, M7‐Luc, PBMC) using Env‐pseudotyped viruses and Env.IMC.LucR viruses. Binding antibodies (Env‐specific total IgG, IgG subclasses, IgA, IgM) are quantified in the systemic and mucosal compartments by using a customized luminex platform. Antibody avidity is measured by using surface plasmon resonance (SPR). We also have capacity to assess FcR‐mediated antibody effector functions, including antibody‐dependent cellular cytotoxicity (ADCC) and antibody‐dependent cell‐mediated viral inhibition (ADCVI). A newly standardized PBMC neutralization assay utilizing Env.IMC.LucR viruses is available for improved assessments of vaccine‐elicited NAbs. Automation of the TZM‐bl assay and Env‐pseudotyped virus production are now available at NVITAL and the HSC, respectively. We have established a large repository of well‐characterized env plasmid DNAs, Env‐pseudoviruses and Env.IMC.LucR viruses, which strengthens our ability to assess vaccine‐elicited neutralizing antibody responses. The Binding Ab and Clinical NAb Assay Cores have implemented a new peptide microarray technology in collaboration with JPT Peptide Technologies that is used to compare vaccine‐elicited antibody responses and to identify peptide binding patterns that associate with potentially protective antibodies. The CA‐VIMC is working to expand its scope by providing new assay technologies for measurements of virion binding, aggregation and phagocytosis, FcR binding affinity, virus migration in mucosal fluids, inhibition of gp120‐α4β7 binding, and by adopting the peptide microarray technology for measurements of IgG1‐4 and IgA. Epitopes for broadly‐reactive neutralizing antibodies are studied by using overlapping peptide arrays, gp120 absorption experiments, trimer binding assays, mutant viruses and computational analyses. These latter assays were used to delineate the epitope specificities of broadly neutralizing antibodies in nine elite neutralizers (collaboration with CHAVI). Finally, in preparation for pending phase 2b trials, the Standard Virus Panel Consortium in the CA‐VIMC is developing an expanded panel of clade C viruses from southern Africa, with an emphasis on transmitted/founder Envs.

One of our highest priorities in the first half of Yr 05 year was to assess the antibody response in RV144 and related clinical trials. Our consortium assessed binding antibodies, neutralizing antibodies, ADCC and ADCVI in non‐case control samples from these trials. Together with the RV144 Humoral/Innate Working Group, the CA‐VIMC also coordinated a study to evaluate and compare the specificity, reproducibility and accuracy of ADCC and ADCVI assays in multiple laboratories. Results from this comparative study were analyzed by the VISC and will be informative when deciding assays for case/control samples from RV144. Finally, we also continued to make progress toward the completion of all assays (>46,000) for the Neutralization Serotype Discovery Project (NSDP). The complete NSDP dataset will be analyzed to decide the optimal choice of reference strains and to seek new insights for the design of novel vaccine immunogens.

 
Submitted July 1, 2010

The Comprehensive Antibody Vaccine Immune Monitoring Consortium (CA‐VIMC) designs and utilizes valid laboratory criteria to judge candidate HIV/AIDS vaccines based on antibody responses in preclinical and clinical trials. We also seek new scientific insights that will help bridge the gap between preclinical vaccine discovery and human clinical trials. Highly standardized and validated assays are used to assess neutralizing antibodies in a GCLP‐compliant manner at multiple domestic and international sites. Binding antibodies (virus‐specific total IgG, IgG subclasses, IgA, IgM) are quantified in serum and mucosal specimens by using a customized luminex platform. Additional core laboratories are performing assays for Fc receptor‐mediate antiviral antibody functions, including antibody‐dependent cellular cytotoxicity (ADCC) and antibody‐dependent cell‐mediated virus inhibition (ADCVI). Epitopes of broadly‐reactive neutralizing antibodies are studied by using overlapping peptide arrays, gp120 absorption experiments, trimer binding assays, mutant virus assays and computational analyses. All of these activities have been strengthened by our cloning and sequencing efforts that have created >600 Env‐pseudotyped viruses and full‐length infectious molecular clones as reference reagents.

Scientifically we have made considerable progress understanding the antigenic diversity of HIV‐1 as it relates to neutralizing antibodies and vaccine development. Over the past year the CA‐VIMC helped delineate the magnitude, breadth and epitope specificity of the neutralizing antibody response in acute and chronic HIV‐1 infection, and to characterize new broadly neutralizing monoclonal antibodies. Our computational analysis of a large sequence and neutralization dataset identified a specific site on the virus (CD4i region of gp120) as a key determinant of broadly neutralizing antibody responses. We are attempting to translate this latter information into novel vaccine concepts. We anticipate gaining additional insights for new vaccine designs as we acquire more information about epitopes that drive the autologous and heterologous neutralizing antibody response in HIV‐1‐infected individuals and in preclinical and clinical vaccine trials. As part of this latter effort, we made considerable progress over the past year in completing a large neutralization serotype discovery project (NSDP) that aims to improve our understanding of the requirements for broadly neutralizing antibody responses.

One of our highest priorities in the second half of this past fiscal year was to prepare for and to conduct studies of the antibody response in the recently‐completed RV144 efficacy trial in Thailand. The modest efficacy seen in this trial affords the first opportunity to identify an immunologic correlate of protection against the acquisition of HIV‐1 infection. A correlate of protection in RV144 would point to the desired type of immune response to elicit with vaccines and would guide the selection of the most promising new vaccine immunogens to test in follow‐up efficacy trials. Our consortium is currently investigating multiple types of antibodies in non‐case control samples from RV144 and related vaccine trials in Thailand. The results will be used to design and conduct a series of experiments on case‐control samples aimed at identifying a correlate of protection.

 
Submitted December 17, 2009 (Interim Report)

The Comprehensive Antibody Vaccine Immune Monitoring Consortium (CA-VIMC) was established to design valid laboratory criteria to judge candidate HIV/AIDS vaccines based on antibody responses in preclinical and clinical stages of testing. The CA-VIMC also aims to generate new scientific findings that will help bridge the gap between preclinical vaccine discovery and human clinical trials. We continue to make substantial progress in enhancing our overall operational efficiency and broadened our collaborations with other CAVD investigators. The CA-VIMC offers highly standardized and validated assays for neutralizing antibodies with high throughput capacity and GCLP compliance at multiple domestic and international laboratories. In addition, the binding antibody core offers standardized HIV-1 and SIV-1 customized luminex assays, IgG purification and quantitative and qualitative validated ELISAs. Our capacity for assessing other antiviral antibody effector functions was expanded to include antibody-dependent cell-mediated viral inhibition (ADCVI). New technologies for measuring antibody-dependent cellular cytotoxicity (ADCC) and inhibition of cell-cell transmission are in development. Moreover, a new PBMC assay technology with high throughput capacity for neutralizing antibodies is now available that utilizes infectious molecular clones carrying a Renilla luciferase reporter gene. Our ability to assess vaccine-elicited neutralizing antibodies and to study broadly neutralizing antibodies has been enhanced by the creation and characterization of over 400 unique Env clones of multiple genetic subtypes from diverse geographic locations. A program for high capacity Env-pseudotyped virus production has been implemented in collaboration with Dr. Hagen von Briesen that enhances the quality and reproducibility of neutralizing antibody assay results across laboratories. To further strengthen our inter-laboratory assay performance, a standardized TZM-bl assay proficiency testing program is fully implemented and open for enrollment.

Scientifically, the CA-VIMC continues to make progress in understanding the antigenic diversity of HIV-1 as it relates to neutralizing antibodies and vaccine development. These efforts involve a variety of sophisticated reagents and new analytical tools for delineating genetic and antigenic features associated with broadly neutralizing antibody responses. Our recent progress includes a study with CHAVI for the extensive investigation and epitope mapping of sera from HIV-1 infected individuals who exhibit high titers of broadly cross-reactive neutralizing antibodies. In addition, we have completed a study that refines the tier classification of 109 molecularly cloned Env-pseudotyped viruses (manuscript in press, J.Virol.). We nearly reached the half-way mark for performance of neutralization assays for the Neutralization Serotype Discovery Project (NSDP), a study in which approximately 40,000 neutralization assays will be performed with over 200 HIV-1 Env-pseudotyped viruses representing all major genetic subtypes and serum from 200 HIV-1 infected individuals representing diverse geographic locations. Computational analysis of the Pilot NSDP dataset was completed in the first half of year 4 and revealed four genetic signatures of broadly neutralizing antibody responses in HIV-infected individuals (manuscript in preparation), thus supporting the larger NSDP. Data from the NSDP will be analyzed in part to decide the optimal choice of reference strains and to seek new information to inform vaccine design.

 
Submitted July 1, 2009

The Comprehensive Antibody Vaccine Immune Monitoring Consortium (CAVIMC) was established to design valid laboratory criteria to judge candidate HIV/AIDS vaccines based on antibody responses in preclinical and clinical stages of testing. It was also established to generate new scientific findings that will help bridge the gap between preclinical vaccine discovery and human clinical trials.  The CAVIMC aims to accelerate the development of an effective HIV/AIDS vaccine by contributing new assay technologies, high quality reagents, proven testing methods and quality assurance oversight consistent with the GHAVE Scientific Strategic Plan for laboratory standardization.  It will expand the size and scope of immune monitoring laboratories to keep pace with vaccines tested by the CAVD and other vaccine discovery groups.  In the case of human clinical trials, all work will be conducted in adherence with Good Clinical Laboratory Practices (GCLP) to further facilitate the licensure and timely access of approved products.  Finally, the CAVIMC trains scientists in developing countries and provides them with financial support with the goal of facilitating international vaccine trials and enabling new scientific initiatives that will lead to more rapid vaccine discovery.

Year 3 was a busy and highly productive year for the CAVIMC.  Our standardized assay services were used heavily by the VDCs, especially for neutralizing antibody assays.  Moreover, our laboratories that measure binding antibodies and ADCC made substantial improvements in methodology, and they contributed to newly published findings on the antibody response in HIV-1-infected individuals.  The quality of work at Core and International Regional Laboratories was strengthened by a highly dedicated Central Quality Assurance Unit.  Substantial progress was made in creating new panels of genetically and antigenically diverse Env-pseudotyped viruses and in producing abundant stocks of these Env¬pseudotyped viruses for use in neutralizing antibody assays by CAVIMC Core and International Regional Laboratories and by other CAVD investigators.  In addition, the Neutralization Serotype Discovery Project was fully mobilized and is on target for completion in the first quarter of 2010.

Scientifically, the CAVIMC made several important contributions in year 3.  Among these was a comprehensive assessment of epitopes recognized by broadly neutralizing plasmas from individuals infected with HIV-1 subtypes B and C, and the development of new strategies and reagents to delineate epitopes that elicit broadly neutralizing antibodies during HIV-1 infection.  Additionally, we devised an improved classification system for defining the neutralization phenotype of reference strains that will provide more meaningful quantitative assessment of vaccine-elicited neutralizing antibody responses.

Finally, a new project was initiated that aims to improve our ability to adequately assess neutralizing antibodies and other potentially protective antibodies using multiple platforms.  In collaboration with other CAVIMC investigators, this project made considerable progress in developing a new PBMC-based assay technology that utilizes infectious molecular clones carrying a Renilla luciferase reporter gene (IMC-rLuc viruses). This new assay technology is gaining momentum and contributed to several CAVIMC activities in year 3. 

 
Submitted January 1, 2009 (Interim Report)

We continue to make substantial progress in enhancing our overall operational efficiency and broadened our collaborations with other CAVD investigators. The CA-VIMC offers highly standardized and validated assays for binding antibodies and neutralizing antibodies with high throughput capacity and GCLP compliance.  Our capacity for assessing other antiviral antibody effector functions was expanded to include antibody-dependent cell-mediated viral inhibition (ADCVI).  New technologies for measuring antibody-dependent cellular cytotoxicity (ADCC) and inhibition of cell-cell transmission are in development.  Moreover, a new PBMC assay technology with high throughput capacity for neutralizing antibodies is now available that utilizes infectious molecular clones carrying a Renilla luciferase reporter gene.  Our ability to assess vaccine-elicited neutralizing antibodies and to study broadly neutralizing antibodies has been enhanced by the creation and characterization of over 330 unique Env clones of multiple genetic subtypes from diverse geographic locations.  A new program for high capacity Env-pseudotyped virus production was initiated in collaboration with Dr. Hagen von Breisen that promises to enhance the quality and reproducibility of neutralizing antibody assay results across laboratories.  To further strengthen our inter-laboratory assay performance, a standardized TZM-bl assay proficiency testing program was finalized and will be implemented in the first quarter of 2009. 

Scientifically, the CA-VIMC continues to make progress in understanding the antigenic diversity of HIV-1 as it relates to neutralizing antibodies and vaccine development.  These efforts involve a variety of sophisticated reagents and new technical and analytical tools for delineating genetic and antigenic features associated with broadly neutralizing antibody responses.  Our recent progress includes the completion of an extensive investigation of the epitope specificity of broadly neutralizing antibodies in serum from clade B and clade C HIV-1-infected individuals (J, Virology, in press).  In addition, we identified clade-related neutralization phenotypes and associated genetic signatures (Virology, in press) and we have improved the definition of tier 1 and tier 2 neutralization phenotypes for 107 Env-pseudotyped viruses (manuscript in preparation).  Moving forward, we recently generated additional large datasets that will be analyzed by agglomerative heirarchical clustering analysis (heat maps) and other statistical methods that we hope will provide new insights for vaccine design and testing.  Also, we are in the final stages of implementing our Neutralization Serotype Discovery Project (NSDP) in which 90,000 neutralization assays will be perfomed in 2009 with 300 HIV-1 viruses and serum from 300 HIV-1 infected individuals representing all major genetic subtypes of the virus.  These data will be analyzed to decide the optimal choice of reference strains for neutralizing antibody assays and to seek additional information to inform vaccine design.

 
Submitted July 1, 2008

The Comprehensive Antibody Vaccine Immune Monitoring Consortium (CA-VIMC) was established to design valid laboratory criteria to judge candidate HIV/AIDS vaccines by the antibody responses they elicit in preclinical and clinical stages of testing. The CA-VIMC aims to speed the development of an effective HIV/AIDS vaccine by contributing new technologies, high quality reagents, proven testing methods and quality assurance oversight consistent with the GHAVE Scientific Strategic Plan for laboratory standardization. In addition, it will expand the size and scope of laboratories to keep pace with vaccines tested by the CAVD and other vaccine discovery groups who are in need of these services. In the case of human clinical trials, all work will be conducted in adherence with Good Clinical Laboratory Practices (GCLP) to further facilitate the licensure and timely access of approved products. Finally, the CA-VIMC aims to train scientists in developing countries and to provide them with financial support with the goal of facilitating international vaccine trials and enabling new scientific initiatives that will lead to more rapid vaccine discovery.

The CA-VIMC became fully operative in this second year of performance, with 27 co-investigators at 20 institutions world-wide. We are providing high quality assay services for neutralizing antibodies, binding antibodies and ADCC. These efforts were strengthened this year by the creation of nearly 100 new full-length functional Env clones by the Standard Virus Panel Consortium (SVPC). Through the efforts of our R&D program, we also have a growing capacity to dissect the epitope specificity of neutralizing antibodies, to determine Env trimer binding and to identify genetic signatures of clade-related neutralization phenotypes. Adding to this capacity, a global network of Regional Laboratories in Africa, Thailand, China, India and South America is becoming competent and proficient in the performance of neutralizing antibodies, where several Regional Laboratories made significant progress toward GLCP compliance. These Regional Laboratories are demonstrating potential for downstream rewards in terms of global preparedness for clinical trials and scientific progress by investigators in developing countries. Finally, we implemented a new program that aims to facilitate a high throughput PBMC/reporter gene assay technology for neutralizing antibodies. 

 
Submitted December 31, 2007 (Interim Report)

Substantial progress was made over the past 6 months that has enhanced our overall operational efficiency and broadened our collaborations with other CAVD investigators.  The CA-VIMC now offers highly standardized and validated assays for binding and neutralizing antibodies with high throughput capacity at Duke and Harvard.  Increased capacity for GCLP neutralizing antibody assays at NVITAL is expected by the end of the third quarter of year 2 and will be used initially to assess Tier 2 neutralizing antibody responses in phase II and III clinical trials of vCP priming/gp120 boosting.  In conjunction with the Operations Core, NVITAL also facilitated the production of GMP plasmid DNAs for pseudoviruse production to be shared with all CAVD investigators.  International participation in the vaccine discovery process was enhanced by the Central Reference Laboratory completing the training of scientists at eight Regional Laboratories and beginning a program to engage the sites in meaningful studies of neutralizing antibodies.  All of these efforts are closely monitored by a fully operational Central Quality Assurance Unit.

The Standard Virus Panel Consortium, which struggled initially from a paucity of specimens, is now in full swing thanks to large numbers of specimens donated by the HMJF, CHAVI, IAVI and individual investigators.  Thus, we are expecting several new panels of reference Env-pseudotyped viruses in the coming year that will cover genetic subtypes for which few reference strains currently exist (e.g., subtypes A, D, G, AE and AG).  Additional progress was made in the development of ancillary assays, including ADCC, inhibition of cell-cell transmission, neutralization assays in macrophages, assays for complement activating antibodies, neutralization in PBMC using infectious molecular clones, and novel assays to map neutralizing antibody epitopes.  The goal is for newer, non-standardized assays to move quickly from a development phase to standardized and validated assays.   A description of all laboratory services may be found at the CA-VIMC CSF site on the CAVD portal.  This site also contains contact information and documents for initiating the study registration process and to view a growing registry of virus panels available for neutralizing antibody testing.

A second round of proficiency testing was completed, and plans are being finalized, to begin the third round of testing in January 2008 in conjunction with the CQAU and VISC.  Analyses of results from the second round indicates inter-laboratory concordance was greater with the use of centrally prepared pseudoviruses.  This third round will likely serve as a validation of the preliminary proficiency testing program.  Once a validated and standardized proficiency testing program is in place, this program will be made available to everyone in the field but will be mandatory for those seeking to serve as GCLP neutralizing antibody labs.  The HSC (von Briesen) has been identified as the site for central preparation and distribution of pseudoviruses for the proficiency testing program and collaborations have been initiated, including completion of training in and comparison assays with the Central Reference Laboratory.

The CA-VIMC, in conjunction with the CAVD Alliance Management Team, initiated an ongoing CAVD-wide collaboration for the manufacture of a set of multi-clade gp140 proteins for the use of the CAVD community.  This initiative united the various VIMCs and VDCs in not only deciding which strains and gp140s to produce but the conformation and manufacturer.  This further sparked innovative discussions and identified areas for future collaborations.      

Finally, two separate meetings (one Site Visit and one Scientific Advisory Board) of CA-VIMC investigators, including Jose Esparza, James Kublin, Rick Koup, Hagen von Briesen and SAB members, were held on October 4, 2007 and October 29-30, 2007 respectively, in Durham, NC.  These meetings were used to evaluate the CA-VIMC program and address and resolve barriers to progress. 

 
Submitted July 1, 2007

The Comprehensive Antibody Vaccine Immune Monitoring Consortium (CA-VIMC) was established to design valid laboratory criteria to judge candidate HIV/AIDS vaccines as determined by antibody responses in preclinical and clinical stages of testing.  The CA-VIMC aims to speed the development of an effective HIV/AIDS vaccine by contributing new technologies, high quality reagents, proven testing methods and quality assurance oversight consistent with the GHAVE Scientific Strategic Plan for laboratory standardization.  In addition, it will expand the size and scope of laboratories to keep pace with vaccines tested by the CAVD and other vaccine discovery groups who are in need of these services.  In the case of human clinical trials, this work will be done in strict adherence with Good Clinical Laboratory Practice (GCLP) guidelines to further facilitate the licensure and timely access of approved products.  Finally, the CA-VIMC aims to train scientists in developing countries and to provide them with financial support with the goal of facilitating international vaccine trials and enabling new scientific initiatives that will lead to more rapid vaccine discovery.

The CA-VIMC became fully operational by the end of year 1.  All subcontracts were established except one, which is in final stages of completion.  Immune monitoring services were implemented that have kept pace with the needs of the CAVD Vaccine Discovery Groups.  Moreover, requisite electronic systems for study registration and data acquisition and analysis were designed in coordination with the VISC and are now fully implemented.  Technical and GCLP training of scientists from eight international Regional Laboratories is moving at a rapid pace and promises to extend the capabilities of these laboratories very soon.  Also, considerable progress was made in developing computational tools and an exciting R&D project aimed at delineating key neutralization epitopes for vaccine discovery.  Our progress in developing much-needed reference reagents got off to a slow start in year 1 but measures are being taken to facilitate this process in subsequent years.

 
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