David A. Garber

Prospects for an AIDS vaccine: three big questions, no easy answers

The unremitting devastation created by the AIDS pandemic will probably only be controlled when a vaccine is developed that is safe, effective, affordable, and simple enough to permit implementation in developing countries where the impact of AIDS is most severe. Although formidable practical, political, economic, social, and ethical challenges face the AIDS vaccine development effort, the most fundamental challenges now reside at the level of the basic biology of HIV-1 infection and pathogenesis. Of these biological considerations, three questions loom especially large: can we design immunogens that will elicit neutralising antibodies that are reactive against a wide variety of primary HIV isolates; will vaccine-elicited cytotoxic T cells be fundamentally better at controlling HIV-1 replication and ameliorating disease progression than those responses that arise during natural HIV infection; and to what extent will the tremendous global genetic diversity of HIV-1 compromise the breadth of vaccine-elicited protective immunity and the overall effectiveness of an AIDS vaccine? Although these are three exceptionally challenging questions, they are now being approached with clear hypotheses whose testing is being facilitated by an ever-improving array of technologies for vaccine design and immunological characterisation. The extent to which the field of AIDS vaccine research can now come together to answer these questions in the best coordinated, most efficient manner will probably be an important determinant of how and when an effective AIDS vaccine will be developed.

Selective removal of stratum corneum by microdermabrasion to increase skin permeability

This study sought to determine if microdermabrasion can selectively remove stratum corneum to increase skin permeability. Although, microdermabrasion has been used for cosmetic treatment of skin for decades, no study has assessed the detailed effects of microdermabrasion conditions on the degree of skin tissue removal. Therefore, we histologically characterized the skin of rhesus macaques and human volunteers after microdermabrasion at different conditions. Using mobile tip microdermabrasion, an increase in the number of treatment passes led to greater tissue removal ranging from minimal effects to extensive damage to deeper layers of the skin. Of note, these data showed for the first time that at moderate microdermabrasion conditions selective yet full-thickness removal of stratum corneum could be achieved with little damage to deeper skin tissues. In the stationary mode of microdermabrasion, selective stratum corneum removal was not observed, but micro-blisters could be seen. Similar tissue removal trends were observed in human volunteers. As proof of concept for drug delivery applications, a model fluorescent drug (fluorescein) was delivered through microdermabraded skin and antibodies were generated against vaccinia virus after its topical application in monkeys. In conclusion, microdermabrasion can selectively remove full-thickness stratum corneum with little damage to deeper tissues and thereby increase skin permeability.

Expression of CCL20 and Granulocyte-Macrophage Colony-Stimulating Factor, but Not Flt3-L, from Modified Vaccinia Virus Ankara Enhances Antiviral Cellular and Humoral Immune Responses

ABSTRACT While modified vaccinia virus Ankara (MVA) is currently in clinical development as a safe vaccine against smallpox and heterologous infectious diseases, its immunogenicity is likely limited due to the inability of the virus to replicate productively in mammalian hosts. In light of recent data demonstrating that vaccinia viruses, including MVA, preferentially infect antigen-presenting cells (APCs) that play crucial roles in generating antiviral immunity, we hypothesized that expression of specific cytokines and chemokines that mediate APC recruitment and activation from recombinant MVA (rMVA) vectors would enhance the immunogenicity of these vectors. To test this hypothesis, we generated rMVAs that express murine granulocyte-macrophage colony-stimulating factor (mGM-CSF), human CCL20/human macrophage inflammatory protein 3α (hCCL20/hMIP-3α), or human fms-like tyrosine kinase 3 ligand (hFlt3-L), factors predicted to increase levels of dendritic cells (DCs), to recruit DCs to sites of immunization, or to promote maturation of DCs in vivo, respectively. These rMVAs also coexpress the well-characterized, immunodominant lymphocytic choriomeningitis virus nucleoprotein (NP) antigen that enabled sensitive and quantitative assessment of antigen-specific CD8+ T-cell responses following immunization of BALB/c mice. Our results demonstrate that immunization of mice with rMVAs expressing mGM-CSF or hCCL20, but not hFlt3-L, results in two- to fourfold increases of cellular immune responses directed against vector-encoded antigens and 6- to 17-fold enhancements of MVA-specific antibody titers, compared to those responses elicited by nonadjuvanted rMVA. Of note, cytokine augmentation of cellular immune responses occurs when rMVAs are given as primary immunizations but not when they are used as booster immunizations, suggesting that these APC-modulating proteins, when used as poxvirus-encoded adjuvants, are more effective at stimulating naïve T-cell responses than in promoting recall of preexisting memory T-cell responses. Our results demonstrate that a strategy to express specific genetic adjuvants from rMVA vectors can be successfully applied to enhance the immunogenicity of MVA-based vaccines.

Vaccine-Induced, Simian Immunodeficiency Virus-Specific CD8+ T Cells Reduce Virus Replication but Do Not Protect from Simian Immunodeficiency Virus Disease Progression

Our limited understanding of the interaction between primate lentiviruses and the host immune system complicates the design of an effective HIV/AIDS vaccine. To identify immunological correlates of protection from SIV disease progression, we immunized two groups of five rhesus macaques (RMs) with either modified vaccinia Ankara (MVA) or MVAΔudg vectors that expressed SIVmac239 Gag and Tat. Both vectors raised a SIV-specific CD8+ T cell response, with a magnitude that was greater in mucosal tissues than in peripheral blood. After challenge with SIVmac239, all vaccinated RMs showed mucosal and systemic CD8+ T cell recall responses that appeared faster and were of greater magnitude than those in five unvaccinated control animals. All vaccinated RMs showed a ∼1-log lower peak and early set-point SIV viral load than the unvaccinated animals, and then, by 8 wk postchallenge, exhibited levels of viremia similar to the controls. We observed a significant direct correlation between the magnitude of postchallenge SIV-specific CD8+ T cell responses and SIV viral load. However, vaccinated RMs showed no protection from either systemic or mucosal CD4+ T cell depletion and no improved survival. The observation that vaccine-induced, SIV-specific CD8+ T cells that partially control SIVmac239 virus replication fail to protect from immunological or clinical progression of SIV infection underscores both the complexity of AIDS pathogenesis and the challenges of properly assessing the efficacy of candidate AIDS vaccines.

Expanding the repertoire of Modified Vaccinia Ankara-based vaccine vectors via genetic complementation strategies.

Background Modified Vaccinia virus Ankara (MVA) is a safe, highly attenuated orthopoxvirus that is being developed as a recombinant vaccine vector for immunization against a number of infectious diseases and cancers. However, the expression by MVA vectors of large numbers of poxvirus antigens, which display immunodominance over vectored antigens-of-interest for the priming of T cell responses, and the induction of vector-neutralizing antibodies, which curtail the efficacy of subsequent booster immunizations, remain as significant impediments to the overall utility of such vaccines. Thus, genetic approaches that enable the derivation of MVA vectors that are antigenically less complex may allow for rational improvement of MVA-based vaccines. Principal Findings We have developed a genetic complementation system that enables the deletion of essential viral genes from the MVA genome, thereby allowing us to generate MVA vaccine vectors that are antigenically less complex. Using this system, we deleted the essential uracil-DNA-glycosylase (udg) gene from MVA and propagated this otherwise replication-defective variant on a complementing cell line that constitutively expresses the poxvirus udg gene and that was derived from a newly identified continuous cell line that is permissive for growth of wild type MVA. The resulting virus, MVAΔudg, does not replicate its DNA genome or express late viral gene products during infection of non-complementing cells in culture. As proof-of-concept for immunological ‘focusing’, we demonstrate that immunization of mice with MVAΔudg elicits CD8+ T cell responses that are directed against a restricted repertoire of vector antigens, as compared to immunization with parental MVA. Immunization of rhesus macaques with MVAΔudg-gag, a udg − recombinant virus that expresses an HIV subtype-B consensus gag transgene, elicited significantly higher frequencies of Gag-specific CD8 and CD4 T cells following both primary (2–4-fold) and booster (2-fold) immunizations as compared to the udg + control virus MVA-gag, as determined by intracellular cytokine assay. In contrast, levels of HIV Gag-specific antibodies were elicited similarly in macaques following immunization with MVAΔudg-gag and MVA-gag. Furthermore, both udg − and udg + MVA vectors induced comparatively similar titers of MVA-specific neutralizing antibody responses following immunization of mice (over a 4-log range: 104–108 PFU) and rhesus macaques. These results suggest that the generation of MVA-specific neutralizing antibody responses are largely driven by input MVA antigens, rather than those that are synthesized de novo during infection, and that the processes governing the generation of antiviral antibody responses are more readily saturated by viral antigen than are those that elicit CD8+ T cell responses. Significance Our identification of a spontaneously-immortalized (but not transformed) chicken embryo fibroblast cell line (DF-1) that is fully permissive for MVA growth and that can be engineered to stably express MVA genes provides the basis for a genetic system for MVA. DF-1 cells (and derivatives thereof) constitute viable alternatives, for the manufacture of MVA-based vaccines, to primary CEFs – the conventional cell substrate for MVA vaccines that is not amenable to genetic complementation strategies due to these cells finite lifespan in culture. The establishment of a genetic system for MVA, as illustrated here to allow udg deletion, enables the generation of novel replication-defective MVA mutants and expands the repertoire of genetic viral variants that can now be explored as improved vaccine vectors.

Viral CTL Escape Mutants Are Generated in Lymph Nodes and Subsequently Become Fixed in Plasma and Rectal Mucosa during Acute SIV Infection of Macaques

SIVmac239 infection of rhesus macaques (RMs) results in AIDS despite the generation of a strong antiviral cytotoxic T lymphocyte (CTL) response, possibly due to the emergence of viral escape mutants that prevent recognition of infected cells by CTLs. To determine the anatomic origin of these SIV mutants, we longitudinally assessed the presence of CTL escape variants in two MamuA*01-restricted immunodominant epitopes (Tat-SL8 and Gag-CM9) in the plasma, PBMCs, lymph nodes (LN), and rectal biopsies (RB) of fifteen SIVmac239-infected RMs. As expected, Gag-CM9 did not exhibit signs of escape before day 84 post infection. In contrast, Tat-SL8 escape mutants were apparent in all tissues by day 14 post infection. Interestingly LNs and plasma exhibited the highest level of escape at day 14 and day 28 post infection, respectively, with the rate of escape in the RB remaining lower throughout the acute infection. The possibility that CTL escape occurs in LNs before RBs is confirmed by the observation that the specific mutants found at high frequency in LNs at day 14 post infection became dominant at day 28 post infection in plasma, PBMC, and RB. Finally, the frequency of escape mutants in plasma at day 28 post infection correlated strongly with the level Tat-SL8-specific CD8 T cells in the LN and PBMC at day 14 post infection. These results indicate that LNs represent the primary source of CTL escape mutants during the acute phase of SIVmac239 infection, suggesting that LNs are the main anatomic sites of virus replication and/or the tissues in which CTL pressure is most effective in selecting SIV escape variants.

Deletion of Specific Immune-Modulatory Genes from Modified Vaccinia Virus Ankara-Based HIV Vaccines Engenders Improved Immunogenicity in Rhesus Macaques

ABSTRACT Modified vaccinia virus Ankara (MVA) is a safe, attenuated orthopoxvirus that is being developed as a vaccine vector but has demonstrated limited immunogenicity in several early-phase clinical trials. Our objective was to rationally improve the immunogenicity of MVA-based HIV/AIDS vaccines via the targeted deletion of specific poxvirus immune-modulatory genes. Vaccines expressing codon-optimized HIV subtype C consensus Env and Gag antigens were generated from MVA vector backbones that (i) harbor simultaneous deletions of four viral immune-modulatory genes, encoding an interleukin-18 (IL-18) binding protein, an IL-1β receptor, a dominant negative Toll/IL-1 signaling adapter, and CC-chemokine binding protein (MVAΔ4-HIV); (ii) harbor a deletion of an additional (fifth) viral gene, encoding uracil-DNA glycosylase (MVAΔ5-HIV); or (iii) represent the parental MVA backbone as a control (MVA-HIV). We performed head-to-head comparisons of the cellular and humoral immune responses that were elicited by these vectors during homologous prime-boost immunization regimens utilizing either high-dose (2 × 108 PFU) or low-dose (1 × 107 PFU) intramuscular immunization of rhesus macaques. At all time points, a majority of the HIV-specific T cell responses, elicited by all vectors, were directed against Env, rather than Gag, determinants, as previously observed with other vector systems. Both modified vectors elicited up to 6-fold-higher frequencies of HIV-specific CD8 and CD4 T cell responses and up to 25-fold-higher titers of Env (gp120)-specific binding (nonneutralizing) antibody responses that were relatively transient in nature. While the correlates of protection against HIV infection remain incompletely defined, our results indicate that the rational deletion of specific genes from MVA vectors can positively alter their cellular and humoral immunogenicity profiles in nonhuman primates.

Vaccine-induced T cells Provide Partial Protection Against High-dose Rectal SIVmac239 Challenge of Rhesus Macaques

Despite enormous efforts by the scientific community, an effective HIV vaccine remains elusive. To further address to what degree T cells in absence of antibodies may protect against simian immunodeficiency virus (SIV) disease progression, rhesus macaques were vaccinated intramuscularly with a chimpanzee-derived Ad vector (AdC) serotype 6 and then boosted intramuscularly with a serologically distinct AdC vector of serotype 7 both expressing Gag of SIVmac239. Animals were subsequently boosted intramuscularly with a modified vaccinia Ankara (MVA) virus expressing Gag and Tat of the homologous SIV before mucosal challenge with a high dose of SIVmac239 given rectally. Whereas vaccinated animals showed only a modest reduction of viral loads, their overall survival was improved, in association with a substantial protection from the loss of CD4(+) T cells. In addition, the two vaccinated Mamu-A*01(+) macaques controlled viral loads to levels below detection within weeks after challenge. These data strongly suggest that T cells, while unable to affect SIV acquisition upon high-dose rectal infection, can reduce disease progression. Induction of potent T-cell responses should thus remain a component of our efforts to develop an efficacious vaccine to HIV-1.

Analysis of putative mucosal SHIV susceptibility factors during repeated DMPA treatments in pigtail macaques.

Depot‐medroxyprogesterone acetate (DMPA) has been associated in some studies with increased HIV susceptibility in women. We used a pigtail macaque model to document the effects of repeated DMPA treatments and their potential contribution to increased SHIV susceptibility.

Rectal Application of a Highly Osmolar Personal Lubricant in a Macaque Model Induces Acute Cytotoxicity but Does Not Increase Risk of SHIV Infection

Background Personal lubricant use is common during anal intercourse. Some water-based products with high osmolality and low pH can damage genital and rectal tissues, and the polymer polyquaternium 15 (PQ15) can enhance HIV replication in vitro. This has raised concerns that lubricants with such properties may increase STD/HIV infection risk, although in vivo evidence is scarce. We use a macaque model to evaluate rectal cytotoxicity and SHIV infection risk after use of a highly osmolar (>8,000 mOsm/kg) water-based lubricant with pH of 4.4, and containing PQ15. Methods Cytotoxicity was documented by measuring inflammatory cytokines and epithelial tissue sloughing during six weeks of repeated, non-traumatic lubricant or control buffer applications to rectum and anus. We measured susceptibility to SHIVSF162P3 infection by comparing virus doses needed for rectal infection in twenty-one macaques treated with lubricant or control buffer 30 minutes prior to virus exposure. Results Lubricant increased pro-inflammatory cytokines and tissue sloughing while control buffer (phosphate buffered saline; PBS) did not. However, the estimated AID50 (50% animal infectious dose) was not different in lubricant- and control buffer-treated macaques (p = 0.4467; logistic regression models). Conclusions Although the test lubricant caused acute cytotoxicity in rectal tissues, it did not increase susceptibility to infection in this macaque model. Thus neither the lubricant-induced type/extent of inflammation nor the presence of PQ15 affected infection risk. This study constitutes a first step in the in vivo evaluation of lubricants with regards to HIV transmission.