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Nonhuman Primate AIDS Vaccine Workshop

Marriott Hotel
Bethesda, Maryland
November 12–13, 2008

Meeting Summary

On November 12–13, 2008, in Bethesda, Maryland, the National Institute of Allergy and Infectious Diseases (NIAID) hosted a workshop on the use of nonhuman primates to study AIDS. A goal of the workshop was to have experts provide guidance on how best to invest resources in nonhuman primate models to advance the development of an effective AIDS vaccine. The workshop featured panel presentations, discussions, and the development of priorities for research topics, resources, and reagents.

Introduction

James Bradac, Ph.D., Chief of the Preclinical Research and Development Branch in NIAID’s Division of AIDS (DAIDS), welcomed the workshop participants. He noted the summit meeting of HIV vaccine researchers that took place in March 2008 in the aftermath of the STEP trial. That meeting determined a need to establish nonhuman primate research priorities and a need to integrate clinical research and discovery. Nancy Miller, Ph.D., of DAIDS’ Vaccine and Prevention Research Program, also welcomed the participants. She charged them to develop recommendations for priority nonhuman primate research topics, funding mechanisms, and broad research strategies.

Session 1: Mucosal Infection—Understanding Early Events of SIV Mucosal Route Infection

Panel: Andrew Lackner Ph.D., D.V.M. (leader); Ashley Haase, M.D.; Brandon Keele, Ph.D.; Daniel Douek, M.D., Ph.D.

The first panel focused on mucosal infection, addressing the following topics:

  • The process of acute SIV infection in genital and rectal mucosa
  • Implications of establishment of infection by a limited number of founder viruses
  • Virus replication, activation, and pathogenic events in the mucosa, including breakdown of the mucosal barrier

Dr. Lackner cited current knowledge, such as the variety of target cells and viruses, and variations in trafficking to nodes. Dr. Haase reviewed the early events in sexual mucosal transmission of virus, noting that inhibitory responses can stimulate infection. Expansion of the virus occurs in a matter of days. Dr. Keele reported on studies of nonhuman primates that demonstrated that the founding virus can be identified and genetically sequenced.

Dr. Douek reviewed our understanding of the immunological and structural breakdown of the GI barrier in mucosal surfaces in acute infection. He described events such as the loss of CD4 T-cells, Th17, dendritic cells, macrophages, B-cells, and more. Each of these events offers an opportunity for study. There are many unknowns. In particular, we do not know the sequence of events—what leads to what? Dr. Douek stressed that immune cells in the gut are different from immune cells elsewhere, and the gut usually does not feature inflammation.

Discussion

In discussion, it was noted that we need to determine the half-life of lymphocytes on the mucosal surface. We need to describe the trafficking patterns of migrating cells, and what determines them. We need to understand the differences between individuals. The amount of virus seems to play a role. Low levels offer an opportunity to control the infection. We do not understand the barrier function (epithelium, lumen, etc.). Furthermore, SIV may not operate in the same way that HIV operates. We need a model that can illuminate transmission. Which viruses are selected for transmission in the mucosa? We need to construct a larger research infrastructure to conduct analyses. We need to understand the massive depletion of CD4 T-cells early in infection. Are there facilitators that we might target? We should decide whether a model aims to prevent acquisition or progression. When studying the loss of dendritic cells (as one example) investigators should consider changes in the functions of the cells. They should study how the mucosal barrier breaks down.

Dr. Lackner summarized the discussion by listing the following priorities for research:

Early events in mucosal infection

  • Define differences in transmission between distinct mucosal sites. Define key events during the early “window” of time when infection is limited to the mucosa at the point of transmission.
  • What is the sequence and kinetics of events between the end of localized mucosal infection through breakdown of the mucosal barrier. Why doesn’t breakdown of the mucosal barrier happen in natural hosts? What is the impact of cell-free vs. cell associated exposure on early events in mucosal infection?
  • What is the impact of local inflammation (e.g. vaginal coinfection) on early events in mucosal infection?
  • Determinants of selection for viral transmission at the mucosa
  • What is the basis for viral selection at the mucosa?
  • Are there viral determinants of transmission or is this a stochastic event -- is this a genetic or physiologic selection? What is the effect of dose on viral selection at the mucosa
  • What is the impact of cell-free vs. cell associated exposure on viral selection at the mucosa?>
  • Standardized protocols with regard to dose, stock, etc. at each mucosal site would be desirable.

Basic mucosal immunology

  • Phenotype and function of resident immune cells
  • Trafficking patterns of mucosal leukocytes
  • Kinetics of mucosal leukocytes Supporting infrastructure
  • Single genome analysis
  • Functional genomics/Systems biology
  • High resolution in vivo imaging
  • Nonhuman primates and supporting resources

Session 2: Understanding Nonpathogenic SIV Infection in Natural Host Species

Panel: Guido Silvestri, M.D. (leader); Vanessa Hirsch, D.V.M., Sc.D.; Joern Schmitz, M.D.; Jacob Estes, Ph.D.; Beatrice Hahn, M.D.

The second panel focused on the following questions regarding SIV and natural hosts:

  • What are the mechanisms underlying the AIDS resistance of natural SIV hosts?
  • What are the mechanisms underlying resistance to mother-to-infant transmission in natural hosts?
  • Do different natural hosts have different mechanisms for avoiding the pathogenic effects of SIV infection?
  • What additional natural hosts (other than sooty mangabeys and African green monkeys) can be studied?
  • What would a vaccine or other intervention have to achieve to change the pathogenic nature of SIV (and HIV) infection?

Dr. Silvestri lamented the lack of research on SIV infection in the natural host. For example, there are no consortium-type grants. We do not know why infected hosts do not develop disease and why mother-to-infant transmission does not occur. Dr. Silvestri called for research to learn the host’s “tricks” leading to those results. Such factors might play roles in human vaccine strategies. In particular, we should determine what causes the low immune activation in the nonhuman primate host, how that protects the animal, why low-CCR5 expression occurs, and the role of the adaptive response.

Dr. Hirsch stated that vertical transmission through breastfeeding occurs in some cases. We need to study the immunology in the nonhuman primate infants, and we need to learn the mechanisms of transmission (breastfeeding and otherwise). Dr. Schmitz described studies attempting to elucidate the mechanisms of inductive immune responses in natural hosts. Dr. Estes proposed studies of the different virus/CD4 T-cell relationship in natural hosts, and he suggested that natural host models might point to non-classical approaches. Dr. Hahn noted that a small fraction of SIV-infected chimpanzees will die if infected. Chimpanzees might be an example of a species that is ahead of humans in learning how to co-exist with the infection.

Discussion

It was noted that most transmission during breastfeeding occurs late. There have been no studies of the breast milk and no studies of maternal antibodies. The low levels of CCR5 are present in various species. Investigators should make comparison studies of free virus and cell-associated virus. Dr. Schmitz emphasized that natural hosts with SIV have persistently high levels of viremia. We could be witnessing either an adaptive immune response or an innate immune response. We need to understand not only the response but the regulation of the response.It was suggested that natural hosts might serve better as models for therapeutic interventions than as models for vaccines. Natural hosts have evolved a strategy for co-existing with the virus. For example, they rapidly develop an anti-inflammatory response. If the most effective vaccine-induced responses were to be invoked during the chronic phase, then studies of natural hosts might be beneficial. If, however, the most effective vaccine-induced responses were to occur at a point before breakout from the gut to the periphery, then studies of natural hosts might not be helpful. The natural host model should be used to help understand pathogenesis, not viral replication. Pathogenesis is multifactorial, as is non-pathogenesis. There may be differences among animals. Hence, many animals should be studied.

Dr. Silvestri summarized the discussion by listing the following research priorities:

  • Determine why natural hosts do not develop disease
  • Determine why mother-to-infant transmission does not occur (establish breeding colonies, study newborns, mechanisms, virus types in breast milk)
  • Increase the number of hosts studied, partnering with African institutions
  • Study basic mechanisms to help translate knowledge to yield an AIDS vaccine
  • Increase collaborations among institutions, featuring large project grants and consortia

Session 3: Generation of Protective Cellular and Humoral Immune Responses

Panel: Louis Picker, M.D. (leader); Nancy Haigwood, Ph.D. (leader); David O’Connor, Ph.D.; Norman Letvin, M.D.; Philip Johnson, M.D.; John Mascola, M.D.

The third panel focused on the following issues for the cellular immune response:

  • Understanding the promise and limits of T-cell function in HIV/SIV control
  • Understanding the mechanisms controlling T-cell targeting of HIV/SIV and clonotype selection, and the impact on response efficacy
  • Understanding the ability of innate and semi-innate cellular immune responses to interfere with HIV/SIV replication, and the regulation of these activities

The panel focused on the following issues for protective antibodies:

  • How can nonhuman primate models inform our understanding of the role of antibodies early in the course of infection
  • What key questions can be addressed in nonhuman primate models regarding specificity and level of antibodies necessary to protect from infection or to modulate disease progression
  • Would studies of the antibody repertoire in infected and immunized macaques be informative to address breadth and potency of the response
  • What is the role of the SHIV model in antibody protection studies

Dr. Picker stressed that we have only a vague understanding of how T-cells are regulated. We need to learn which T-cell effector mechanisms can interfere with HIV in vivo. We must develop tools for such in vivo research. Dr. O’Connor added that we lack an understanding of T-cell specificities and how T-cells interact, often relying on surrogates. The magnitude of T-cell expansion appears to play a key role in events. We need to determine which alleles restrict epitope-specific responses.

Dr. Haigwood stressed the benefits of neutralizing antibodies in viral control. Yet the mechanisms (and kinetics, etc.) of neutralizing antibodies are poorly understood. Dr. Johnson noted that we have not determined the levels of antibody that are protective. We also do not know how to effectively elicit protective antibodies. A gene-transfer approach in animals has found some success.

Discussion

It was noted that the behaviors in natural hosts might provide lessons for changing the mucosal microenvironment and affecting the adaptive immune response. A recent study found that T-cells that produce chemokines are resistant to HIV infection. Perhaps we could elicit such self-protective T-cells. We need sophisticated computational tools to determine what a vaccine must do. Perhaps investigators should pursue simple questions or algorithms about infecting CD4 cell lines. We need to develop predictive values about T-cells in the models. We need to recognize epitopes and a hierarchy. We need better assays and better tools for studying CD4 depletion.

Dr. Picker summarized the discussion of T-cells, listing the following research areas:

Cellular Immunity and Protection from HIV/AIDS

  • T cell function and HIV/SIV control –- Knowing what’s works, what doesn’t, and the means by which efficacious responses can be generated and maintained with vaccination
  • Targeting HIV/SIV: Understanding how the mechanisms governing T cell recognition of HIV/SIV and the selection of the HIV/SIV-specific TCR repertoire during memory/effector development impact of the effectiveness of HIV/SIV immune responses
  • Innate and semi-innate cellular immunity -- Identification of mechanisms with anti-HIV/SIV capability and the regulation of such responses

“Tailoring” interrogation of the landscape to crucial questions in HIV vaccine development

  1. Determine which T cell effector mechanisms are capable of interfering with HIV/SIV replication and latency in vivo (as well as those that might potentiate infection).
  2. Characterize where such protective mechanisms fit in the wide spectrum of T cell function and behavior,
  3. Understand the mechanisms by the appropriate functional subsets are generated, and
  4. Understand the mechanisms controlling the behavior and longevity of protective T cell subsets, such that protective cells are available at or recruitable to the appropriate place and time in sufficient numbers.

Dr. Haigwood summarized the discussion of antibodies, listing the following priorities:

Major areas for future investigation: expansion of B cell findings to date

  • Expanded analyses of the antibody repertoire in macaques infected with SIV/SHIV
  • Mechanisms for maintenance of B cell health
  • Mechanisms by which antibodies enhance B cell responses
  • Mechanisms of neutralization/viral clearance

In vivo studies as part of passive transfer studies:

  • Role of different types of antibodies (neutralizing activity, avidity, ADCC, ADCVI, mucosal, IgA, IgG etc.)
  • Window of opportunity for intervention with passive studies; all studies to date point to the first hours of infection
  • Understanding of the composition and complexity of the “IVIG” and comparisons with pools of mAbs
  • In vivo approaches such as antibody gene transfer

In vivo studies as part of vaccine studies:

  • Support for novel immunogen design to effectively elicit antibody responses
    • Vector and Antigen design and testing (with adjuvants)
    • Modeling and structural work, iterative designs
  • Appropriate animal models for immunogenicity studies; measures of types of immunity elicited (mucosal, systemic)
  • Mapping of epitopes to which vaccines direct responses, compared with natural infection
  • Combined T and B cell vaccine approaches—obvious place for synergies with shared challenge studies, but only if well coordinated and planned

Resource and reagent needs:

  • Development of shared homologous and heterologous SHIV and SIV challenge stocks for cross-protection studies
  • Development of challenge models that approximate quasispecies
  • Continued development of monoclonals with improved B cell antigen sorting, better screening
  • Continued support for reference labs, qualified assays
  • Support for adjuvant development to enhance magnitude, locale, and persistence of immunity

Session 4: Mucosal Immunity—Investigation of Vaccine-Induced Immune Responses at Mucosal Sites

Panel: R. Paul Johnson, M.D. (leader); Michael Murphey-Corb, Ph.D.; Daniel Barouch, M.D., Ph.D.; Genoveffa Franchini, M.D.

The fourth panel focused on the following questions/topics:

  • Are mucosal immune responses needed for an effective AIDS vaccine
  • Is mucosal immunization needed for generation of mucosal responses
  • Can mucosal adjuvants enhance antibody responses at mucosal sites
  • What are the differences in mucosal immune responses generated by immunization with different vaccine approaches? What responses should be measured
  • Can innate immunity reduce the risk of infection

Dr. Johnson noted that mucosal immunity is important for transmission and AIDS pathogenesis. Compartmentalization of the systemic and mucosal immune system is not clear-cut. Mucosal immunity can differ at different sites. We have yet to understand the responses that occur in these complex environments. An immune response (humoral or cellular) at a mucosal site does not necessarily qualify as mucosal immunity. Mucosal sites are fragile, and measurements may perturb the system. Dr. Barouch spoke of the comparative capacities of systems and the need for mucosal challenge studies. We need to understand the degree of compartmentalization following vaccination and the mechanisms of inducing mucosal immunity. Dr. Franchini stressed a need to study the founder virus in the first 3 or 4 days of exposure. We need to determine which T-cell responses to measure for the founder effector cells.

Discussion

It was suggested that sampling peripheral blood does not capture enough of what is happening. We must sample the mucosa. We need reliable assays. One research strategy would be to differentiate how one infection takes and how another infection does not take (the latter is more common). We should perform mucosal challenge studies with various viruses. This would require standardization of various challenge stocks.

Many issues remain undetermined: expansion in the gut, longevity of the immune response, and complexity of T-cells in the mucosa. And non-neutralizing antibodies might play a protective role. NK cells certainly are important.

Dr. Johnson summarized the discussion by listing the following research priorities:

  • Does mucosal vaccination result in improved protection compared with systemic vaccination?
  • Head-to-head comparison of single high dose vs multiple “low” dose mucosal challenge of vaccinated macaques
  • Direct comparison of the ability of different vaccine candidates to induce mucosal immune responses and protection against mucosal challenge
  • Establish reliable models of localized SIV-specific mucosal immunity and protection; induction of these responses by vaccination
  • Comparison of different prime/boost strategies (systemic/mucosal) for ability to induce systemic and mucosal immune responses and protection against mucosal challenge

Reagents/Enabling Technologies–Mucosal Immunity

  • Development of novel approaches to identify protective cellular and humoral mucosal immune responses
  • Establishment of standardized protocols and reagents for analysis of mucosal humoral and cellular immune responses
  • Creation of new reagents:
      – macaque sIgA
      – expanded MHC class I & II tetramers
      – analysis of innate immunity
  • Provision of well-characterized and titered stocks for mucosal challenges

Session 5: Evaluation of Virus/Hose Models

Panel: Jeffrey Lifson, M.D. (leader); Shiu-Lok Hu, Ph.D.; Mark Lewis, Ph.D.; Ronald Desrosiers, Ph.D.; Cristian Apetrei, M.D., Ph.D.; Malcolm Martin, M.D.; David Watkins, Ph.D.

The fifth panel focused on the following topics:

  • The roles, strengths, and weaknesses of different macaque species and subspecies in vaccine research
  • The roles, strengths, and weaknesses of existing SIVs and the potential utility of other SIVs, including primary isolates
  • The roles, strengths, and weaknesses of existing SHIVs
  • The rationales for different applications (challenge routes and modes)

Dr. Hu stressed the importance of identifying monkey models that recapitulate the sequelae of infection and are predictive of human disease. Yet we can learn from all models. We need more comparative studies in macaque species. Different species have different susceptibilities to viral infection. Dr. Lewis noted that pig-tailed macaques tend to be unavailable and are not practical for vaccine studies. A majority of studies today use the Indian rhesus macaque. They seem to be the best model for study but are becoming expensive. Chinese rhesus macaques are becoming more available. They have good utility, and while they have peak viral loads lower than those of Indian rhesus macaques following challenge, they are useful for studying immune responses.

Dr. Desrosiers noted minor drawbacks in the use of SIV rhesus monkey models. He concluded that they are good models nevertheless, showing CD4 depletion, a chronic disease course, a good time frame, and more.

Discussion

The participants agreed that Chinese rhesus macaques seem to be appropriate, with some limitations, as research models for current and new viruses. Acquisition of virus in the Indian and Chinese macaques is similar, although long-term follow-up can differ.

Dr. Lifson summarized the discussion by listing the following research priorities:

  • Continue to use Indian rhesus macaques as model
  • Improve SHIVs
  • Apply virus resources selectively, for example, using informatics data on sequences to inform study strategies
  • Develop new challenge routes, including head-to-head low-dose versus high-dose studies

Dr. Lifson listed the following research priorities within three challenge system components—macaque types, virus options, dosing/frequency:

  • Use different macaque species and viruses to gain insights into what a vaccine can do
  • Make available more monkey species and systems for more efficient use
  • Create synthetic swarms of viral clones of defined properties
  • Establish a curated SIV sequence database (perhaps within Los Alamos)
  • Improve SHIV systems
  • Create challenge models for studying heterologous protection
  • Develop prospective head-to-head data, with a specific challenge virus, comparing high-dose and repeated low-dose mucosal challenges
  • Mimic circulating HIV strains with passaged SIVs

Dr. Lifson proposed that persons with appropriate expertise convene to determine what challenge virus should be used in the head-to-head comparison of high-dose and repeated low-dose mucosal challenges. The experiments should include standardization of stock sequence characterization.

Session 6: Resources and Reagents

Panel: Nancy Miller, Ph.D.; Joern Schmitz, M.D.; Jonathan Warren, Ph.D.; John Harding, Ph.D.; David Watkins, Ph.D.; David Montefiori, Ph.D.; Robert Palermo, Ph.D.; Brandon Keele, Ph.D.; Luis Giavedoni, Ph.D.; Ruth Ruprecht, M.D., Ph.D.

The sixth panel focused on resources and reagents, especially NIH funding resources and assay support for studies.

Funding Resources

Dr. Miller described NIAID resource contracts at three sites, each with BSL-2 capabilities with a total cage capacity of 500–600 animals. Dr. Schmitz described the DAIT contract support for nonhuman primate reagents. It provides antibodies for in vitro and in vivo work, cell lines and transforming viruses, immunoglobin reference reagents, and a database of commercial antibodies. Rhesus-specific antibodies currently are being developed.

Dr. Warren reviewed the DAIDS reagent contract for AIDS vaccine development. The contractor is Quality Biological, Inc., and it provides flexible, targeted resources, including large reagent lots. Dr. Harding described the NCRR-supported National Primate Centers program, which has about 17,000 monkeys, mostly Indian rhesus macaques. About 63 percent of them are in breeding colonies, and there is very little reserve. Dr. Harding cited a need to determine the types of animals that will be needed in the future, a need to identify new sources for monkeys, and a need to develop a strategy regarding Chinese macaques.

Discussion

It was noted that the quality of the animals is important and must be considered. To address the growing problem with numbers, we should develop strategies to use the animal models more efficiently through coordination, reuse, and the application of informatics.

Assay Resources

Dr. Watkins described the service of MHC testing. The service offers high-throughput MHC class 1 genotyping

Dr. Montefiori reviewed the program for neutralizing antibody assays at Duke University. Using a contract mechanism, the program has provided assays for about 200 studies (about 16,000 samples) during the past 6 years. The program uses assays that are sensitive, quantitative, and reproducible, with correlative value and high throughput. Its lead assay is TZM-bl, which is widely used and validated. The program currently is studying this assay to determine whether it fails to detect some neutralizing antibodies. It is addressing other issues such as transitioning to SIV and variabilities in PBMC donors.

Dr. Palermo described the system of microarray analysis of expressed RNA and a program to apply systems biology and global gene expression profiling. This can support rational design of therapeutics and vaccines. Dr. Palermo stated that functional analysis tools are improving. Species-specific tools are still a long way off.

Dr. Keele made a recommendation to develop a single genome applications (SGA) program, with a core facility and a contract mechanism. It might also feature a database with annotation. The program could go beyond the identification of viruses to a determination of what happens over time.

Dr. Giavedoni described luminex assays for cytokine detection at the NCRR-supported National Primate Center. The program validates against seven rhesus monkey cytokines. It requires 25–50 microliters of sample for each test and uses a variety of reagents.

Dr. Ruprecht described the R5 SHIV vaccine challenge strains, which model HIV-1 transmission. They can be used as tools to assess anti-HIV-1 neutralizing antibody response. The 5 SHIVs are in clades B and C. It would be helpful to develop a SHIV in clade A.

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Last Updated August 26, 2009