Timothy Dudek

Rapid Evolution of HIV-1 to Functional CD8+ T Cell Responses in Humanized BLT Mice

Humanized BLT mice accurately develop human HIV-specific CD8+ T cell responses capable of rapidly selecting for CTL escape mutations. Mirror, Mirror One limitation of using animal models of disease is that there’s no magic mirror to tell you which one best reflects human disease. Instead, most animal disease models mimic some aspects of the human condition, but may not recapitulate the disease in its entirety. This limitation is especially true for HIV infection because the virus does not naturally infect mice—the model of choice for biomedical research. Attempts to “humanize” immunodeficient mice through grafting of human immune cells may reconfigure the mouse from a distorting funhouse mirror into a well-lit vanity one. Now, Dudek et al. use humanized BLT (brain, liver, thymus) mice to study human immune responses to HIV. The authors found that HIV-1–specific immune responses in BLT mice mimicked those in humans in terms of specificity, kinetics, and dominant target. Importantly, HIV adapted to the immune responses in these mice just as it does in humans, evolving rapidly to escape from the selective pressure. Indeed, an HLA allele that is protective in humans induced similar protective immune responses in these mice. Although no animal model may perfectly reflect human disease, for HIV infection, humanized BLT mice may be one of the fairest of them all. The development of mouse/human chimeras through the engraftment of human immune cells and tissues into immunodeficient mice, including the recently described humanized BLT (bone marrow, liver, thymus) mouse model, holds great promise to facilitate the in vivo study of human immune responses. However, little data exist regarding the extent to which cellular immune responses in humanized mice accurately reflect those seen in humans. We infected humanized BLT mice with HIV-1 as a model pathogen and characterized HIV-1–specific immune responses and viral evolution during the acute phase of infection. HIV-1–specific CD8+ T cell responses in these mice were found to closely resemble those in humans in terms of their specificity, kinetics, and immunodominance. Viral sequence evolution also revealed rapid and highly reproducible escape from these responses, mirroring the adaptations to host immune pressures observed during natural HIV-1 infection. Moreover, mice expressing the protective HLA-B*57 allele exhibited enhanced control of viral replication and restricted the same CD8+ T cell responses to conserved regions of HIV-1 Gag that are critical to its control of HIV-1 in humans. These data reveal that the humanized BLT mouse model appears to accurately recapitulate human pathogen–specific cellular immunity and the fundamental immunological mechanisms required to control a model human pathogen, aspects critical to the use of a small-animal model for human pathogens.

PD-1 Blockade in Chronically HIV-1-Infected Humanized Mice Suppresses Viral Loads

An estimated 34 million people are living with HIV worldwide (UNAIDS, 2012), with the number of infected persons rising every year. Increases in HIV prevalence have resulted not only from new infections, but also from increases in the survival of HIV-infected persons produced by effective anti-retroviral therapies. Augmentation of anti-viral immune responses may be able to further increase the survival of HIV-infected persons. One strategy to augment these responses is to reinvigorate exhausted anti-HIV immune cells present in chronically infected persons. The PD-1-PD-L1 pathway has been implicated in the exhaustion of virus-specific T cells during chronic HIV infection. Inhibition of PD-1 signaling using blocking anti-PD-1 antibodies has been shown to reduce simian immunodeficiency virus (SIV) loads in monkeys. We now show that PD-1 blockade can improve control of HIV replication in vivo in an animal model. BLT (Bone marrow-Liver-Thymus) humanized mice chronically infected with HIV-1 were treated with an anti-PD-1 antibody over a 10-day period. The PD-1 blockade resulted in a very significant 45-fold reduction in HIV viral loads in humanized mice with high CD8+ T cell expression of PD-1, compared to controls at 4 weeks post-treatment. The anti-PD-1 antibody treatment also resulted in a significant increase in CD8+ T cells. PD-1 blockade did not affect T cell expression of other inhibitory receptors co-expressed with PD-1, including CD244, CD160 and LAG-3, and did not appear to affect virus-specific humoral immune responses. These data demonstrate that inhibiting PD-1 signaling can reduce HIV viral loads in vivo in the humanized BLT mouse model, suggesting that blockade of the PD-1-PD-L1 pathway may have therapeutic potential in the treatment of patients already infected with the AIDS virus.

BLT-humanized C57BL/6 Rag2−/−γc−/−CD47−/− mice are resistant to GVHD and develop B- and T-cell immunity to HIV infection

The use of C57BL/6 Rag2(-/-)γc(-/-) mice as recipients for xenotransplantation with human immune systems (humanization) has been problematic because C57BL/6 SIRPα does not recognize human CD47, and such recognition is required to suppress macrophage-mediated phagocytosis of transplanted human hematopoietic stem cells (HSCs). We show that genetic inactivation of CD47 on the C57BL/6 Rag2(-/-)γc(-/-) background negates the requirement for CD47-signal recognition protein α (SIRPα) signaling and induces tolerance to transplanted human HSCs. These triple-knockout, bone marrow, liver, thymus (TKO-BLT) humanized mice develop organized lymphoid tissues including mesenteric lymph nodes, splenic follicles and gut-associated lymphoid tissue that demonstrate high levels of multilineage hematopoiesis. Importantly, these mice have an intact complement system and showed no signs of graft-versus-host disease (GVHD) out to 29 weeks after transplantation. Sustained, high-level HIV-1 infection was observed via either intrarectal or intraperitoneal inoculation. TKO-BLT mice exhibited hallmarks of human HIV infection including CD4(+) T-cell depletion, immune activation, and development of HIV-specific B- and T-cell responses. The lack of GVHD makes the TKO-BLT mouse a significantly improved model for long-term studies of pathogenesis, immune responses, therapeutics, and vaccines to human pathogens.

Comparison of immunogenicity and protective efficacy of genital herpes vaccine candidates herpes simplex virus 2 dl5-29 and dl5-29-41L in mice and guinea pigs.

A replication-defective herpes simplex virus (HSV)-2 vaccine, dl5-29, which is deleted for two essential early genes, UL5 and UL29, is highly immunogenic and protective in mice and guinea pigs. In a prior study, a derivative of HSV-2 dl5-29 termed dl5-29-41L, which has an additional deletion in UL41 (that encodes the virion-host shut-off protein), was more immunogenic and protective against challenge with wild-type HSV-2 in mice when compared with dl5-29. To determine if deletion of UL41 improves the efficacy of dl5-29 in protecting guinea pigs from HSV-2, animals were immunized with dl5-29, dl5-29-41L, or PBS. The geometric mean neutralizing antibody titers from the dl5-29 and dl5-29-41L recipients were comparable (10(1.97) and 10(2.19), respectively, p=0.15). After intravaginal challenge with wild-type HSV-2, the dl5-29-41L and dl5-29 recipients shed similar titers of HSV-2 from the vagina. Mean acute disease severity scores, numbers of recurrences during 3 months after infection, and latent viral loads in sacral ganglia were similar for dl5-29 and dl5-29-41L (all p values >0.05). dl5-29 and dl5-29-41L completely protected mice from lethal challenge with HSV-2 and induced virus-specific CD8(+) T cells in the spleens of the animals. Thus, dl5-29 was as immunogenic and protective as dl5-29-41L under these conditions. dl5-29 was at least 250,000-fold less virulent than parental virus by intracranial inoculation in healthy mice, and caused no disease in SCID mice. Both dl5-29-41L and dl5-29 are equally effective and immunogenic in guinea pigs, and dl5-29 is very safe in immunocompromised animals.

Evidence for Differences in Immunologic and Pathogenesis Properties of Herpes Simplex Virus 2 Strains From the United States and South Africa

BACKGROUND Genital infection with herpes simplex virus 2 (HSV-2) is linked to an increased risk of infection with human immunodeficiency virus (HIV) in areas such as Sub-Saharan Africa. Thus, an effective genital herpes vaccine would be an important weapon in the fight against HIV/AIDS. METHODS To test whether a current vaccine candidate can protect against HSV-2 from Sub-Saharan Africa, we examined the ability of an HSV-2 vaccine strain, dl5-29, and other HSV-2 replication-defective mutant strains to protect against genital challenge with US or South African strains in a murine model. RESULTS Immunization with dl5-29 reduces infection by both viruses but is significantly more efficacious against the US virus than against the African virus. Furthermore, another US vaccine strain was more efficacious against US than against African viruses, and the converse was observed for the parallel African vaccine strain. Nevertheless, protection against the African viruses was significantly less with all vaccines used in this study. CONCLUSIONS We conclude that there may be differences in protective epitopes and pathogenesis between the US and African strains that raise the need for increased doses of the existing vaccine candidate or an HSV-2 vaccine strain based on viruses from that region.

Construction and properties of a herpes simplex virus 2 dl5-29 vaccine candidate strain encoding an HSV-1 virion host shutoff protein.

The replication-defective herpes simplex virus 2 (HSV-2) dl5-29 mutant virus strain with deletions in the U(L)5 and U(L)29 genes has been shown to protect mice and guinea pigs against challenge with wild-type (wt) HSV-2 and to protect against ocular disease caused by HSV-1 infection. The dl5-29 strain is currently being prepared for clinical trials as a herpes vaccine candidate. As a possible approach to improve the efficacy of dl5-29 as a genital herpes vaccine, we replaced the U(L)41 gene encoding the virion host shutoff function (vhs) with the U(L)41 gene from HSV-1. While the HSV-2 U(L)41 and HSV-1 U(L)41 gene products have analogous functions, vhs-1 is 40-fold less active than vhs-2. Previously, it was shown that disruption of the U(L)41 gene can increase the efficacy of dl5-29 as a vaccine against HSV-2. These properties led us to hypothesize that replacement of vhs-2 by vhs-1 would decrease cytopathic effects in infected host cells, allowing longer survival of antigen-presenting cells and induction of stronger immune responses. The new recombinant dl5-29-41.1 virus shows nearly the same immunogenicity and protection against HSV-2 challenge as the parental dl5-29 virus or a triply deleted mutant virus, dl5-29-41, in the murine model of infection, and grows to higher titers than the parental strain in complementing cells, which is important for GMP production. The results have implications for the design of future HSV-2 vaccine candidates and mechanisms of induction of protective immunity against genital herpes.

Genetic engineering of a modified herpes simplex virus 1 vaccine vector

The herpes simplex virus 1 (HSV-1) d106 mutant virus is a multiple immediate-early gene deletion mutant virus that has been effective as an AIDS vaccine vector in rhesus macaques (Kaur A, Sanford HB, Garry D, Lang S, Klumpp SA, Watanabe D, et al. Ability of herpes simplex virus vectors to boost immune responses to DNA vectors and to protect against challenge by simian immunodeficiency virus. Virology 2007;357:199-214). Further analysis of this vector is needed to advance development into clinical trials. In this study we have defined the precise nature of the multiple IE gene mutations in the d106 viral genome and have used this information to construct a new transfer plasmid for gene transfer into d106. We tested the effect of an additional mutation in the U(L)41 gene on d106 immunogenicity and found that it did not improve the efficacy of the d106 vector, in contrast with results from other studies with U(L)41 gene mutants. The safety profile of d106 was improved by generating a new vector strain, d106S, with increased sensitivity to acyclovir. Finally, we have constructed a d106S recombinant vector that expresses the HIV clade C envelope protein. The d106S HIVenvC recombinant has retained the sensitivity to acyclovir, indicating that this phenotype is a stable property of the d106S vector.

IL-21 induces antiviral microRNA-29 in CD4 T cells to limit HIV-1 infection

Initial events after exposure determine HIV-1 disease progression, underscoring a critical need to understand host mechanisms that interfere with initial viral replication. Although associated with chronic HIV-1 control, it is not known whether interleukin-21 (IL-21) contributes to early HIV-1 immunity. Here we take advantage of tractable primary human lymphoid organ aggregate cultures to show that IL-21 directly suppresses HIV-1 replication, and identify microRNA-29 (miR-29) as an antiviral factor induced by IL-21 in CD4 T cells. IL-21 promotes transcription of all miR-29 species through STAT3, whose binding to putative regulatory regions within the MIR29 gene is enriched by IL-21 signalling. Notably, exogenous IL-21 limits early HIV-1 infection in humanized mice, and lower viremia in vivo is associated with higher miR-29 expression. Together, these findings reveal a novel antiviral IL-21-miR-29 axis that promotes CD4 T-cell-intrinsic resistance to HIV-1 infection, and suggest a role for IL-21 in initial HIV-1 control in vivo.

HIV-specific CD8⁺ T-cell immunity in humanized bone marrow-liver-thymus mice.

CD8(+) T-cell responses play a critical role in the control of human immunodeficiency virus (HIV) infection, and recent vaccine studies in nonhuman primates now demonstrate the ability of T cells to prevent the early dissemination of simian immunodeficiency virus and perhaps clear residual infection. Recent advances in humanized mouse models, in particular the humanized bone marrow-liver-thymus (BLT) mouse model, show promise in their ability not only to support sustained infection with HIV, but also to recapitulate human HIV-specific immunity. The availability of a small-animal model with which to study human-specific immune responses to HIV would greatly facilitate the elucidation of mechanisms of immune control, as well as accelerate the iterative testing of promising vaccine candidates. Here we discuss data from our recent study detailing the composition and efficacy of HIV-specific CD8(+) T-cell responses in humanized BLT mice that was recently presented at a Harvard Center for AIDS Research symposium on humanized mouse models for HIV vaccine design.

Rapid Evolution of HIV-1 to Functional CD8+ T-cell rResponses in Humanized BLT Mice

Results During the acute phase of infection BLT mice rapidly mounted multiple HIV-1-specific CD8+ cellular immune responses against normally immunodominant human CD8 epitopes, with rapid and reproducible viral escape observed within epitopes that similarly tend to escape early in humans. CD8+ T cell responses and viral escape to these same epitopes was confirmed in mice reconstituted with distinct human tissue but expressing the same restricting HLA alleles. Importantly, in two independent groups of mice expressing HLA-B*57 we observed the rapid induction of CD8+ T-cell responses against a number of B*57-restricted responses at frequencies similar to those seen in humans, including the normally immunodominant B*57-IW9, -KF11 and -TW10 epitopes in Gag from which the virus failed to rapidly escape. As in humans, the presence of these conserved responses correlated with significantly greater control over early viral replication. Preliminary vaccine studies in BLT mice support the ability of conventional approaches to induce CD8+ T cell responses and suppress viral loads.