Shawn P. O'Neil

Control of a mucosal challenge and prevention of AIDS by a multiprotein DNA/MVA vaccine

Heterologous prime/boost regimens have the potential for raising high levels of immune responses. Here we report that DNA priming followed by a recombinant modified vaccinia Ankara (rMVA) booster controlled a highly pathogenic immunodeficiency virus challenge in a rhesus macaque model. Both the DNA and rMVA components of the vaccine expressed multiple immunodeficiency virus proteins. Two DNA inoculations at 0 and 8 weeks and a single rMVA booster at 24 weeks effectively controlled an intrarectal challenge administered 7 months after the booster. These findings provide hope that a relatively simple multiprotein DNA/MVA vaccine can help to control the acquired immune deficiency syndrome epidemic.

Nonpathogenic SIV Infection of Sooty Mangabeys Is Characterized by Limited Bystander Immunopathology Despite Chronic High-Level Viremia

HIV-infected humans and SIV-infected rhesus macaques who remain healthy despite long-term infection exhibit exceptionally low levels of virus replication and active antiviral cellular immune responses. In contrast, sooty mangabey monkeys that represent natural hosts for SIV infection do not develop AIDS despite high levels of virus replication and limited antiviral CD8(+) T cell responses. We report here that SIV-infected mangabeys maintain preserved T lymphocyte populations and regenerative capacity and manifest far lower levels of aberrant immune activation and apoptosis than are seen in pathogenic SIV and HIV infections. These data suggest that direct consequences of virus replication alone cannot account for progressive CD4(+) T cell depletion leading to AIDS. Rather, attenuated immune activation enables SIV-infected mangabeys to avoid the bystander damage seen in pathogenic infections and protects them from developing AIDS.

Critical Role for Env as well as Gag-Pol in Control of a Simian-Human Immunodeficiency Virus 89.6P Challenge by a DNA Prime/Recombinant Modified Vaccinia Virus Ankara Vaccine

ABSTRACT Cellular immune responses against epitopes in conserved Gag and Pol sequences of human immunodeficiency virus type 1 have become popular targets for candidate AIDS vaccines. Recently, we used a simian-human immunodeficiency virus model (SHIV 89.6P) with macaques to demonstrate the control of a pathogenic mucosal challenge by priming with Gag-Pol-Env-expressing DNA and boosting with Gag-Pol-Env-expressing recombinant modified vaccinia virus Ankara (rMVA). Here we tested Gag-Pol DNA priming and Gag-Pol rMVA boosting to evaluate the contribution of anti-Env immune responses to viral control. The Gag-Pol vaccine raised frequencies of Gag-specific T cells similar to those raised by the Gag-Pol-Env vaccine. Following challenge, these rapidly expanded to counter the challenge infection. Despite this, the control of the SHIV 89.6P challenge was delayed and inconsistent in the Gag-Pol-vaccinated group and all of the animals underwent severe and, in most cases, sustained loss of CD4+ cells. Interestingly, most of the CD4+ cells that were lost in the Gag-Pol-vaccinated group were uninfected cells. We suggest that the rapid appearance of binding antibody for Env in Gag-Pol-Env-vaccinated animals helped protect uninfected CD4+ cells from Env-induced apoptosis. Our results highlight the importance of immune responses to Env, as well as to Gag-Pol, in the control of immunodeficiency virus challenges and the protection of CD4+ cells.

Differential CD4+ T-Lymphocyte Apoptosis and Bystander T-Cell Activation in Rhesus Macaques and Sooty Mangabeys during Acute Simian Immunodeficiency Virus Infection

ABSTRACT In contrast to pathogenic lentiviral infections, chronic simian immunodeficiency virus (SIV) infection in its natural host is characterized by a lack of increased immune activation and apoptosis. To determine whether these differences are species specific and predicted by the early host response to SIV in primary infection, we longitudinally examined T-lymphocyte apoptosis, immune activation, and the SIV-specific cellular immune response in experimentally infected rhesus macaques (RM) and sooty mangabeys (SM) with controlled or uncontrolled SIV infection. SIVsmE041, a primary SIVsm isolate, reproduced set-point viremia levels of natural SIV infection in SM but was controlled in RM, while SIVmac239 replicated to high levels in RM. Following SIV infection, increased CD8+ T-lymphocyte apoptosis, temporally coinciding with onset of SIV-specific cellular immunity, and elevated plasma Th1 cytokine and gamma interferon-induced chemokine levels were common to both SM and RM. Different from SM, SIV-infected RM showed a significantly higher frequency of peripheral blood activated CD8+ T lymphocytes despite comparable magnitude of the SIV-specific gamma interferon enzyme-linked immunospot response. Furthermore, an increase in CD4+ and CD4−CD8− T-lymphocyte apoptosis and plasma tumor necrosis factor-related apoptosis-inducing ligand were observed only in RM and occurred in both controlled SIVsmE041 and uncontrolled SIVmac239 infection. These data suggest that the “excess” activated T lymphocytes in RM soon after SIV infection are predominantly of non-virus-specific bystander origin. Thus, species-specific differences in the early innate immune response appear to be an important factor contributing to differential immune activation in natural and nonnatural hosts of SIV infection.

Rapid CD4+ T-Cell Loss Induced by Human Immunodeficiency Virus Type 1NC in Uninfected and Previously Infected Chimpanzees

ABSTRACT To investigate the pathogenicity of a virus originating in a chimpanzee with AIDS (C499), two chimpanzees were inoculated with a plasma-derived isolate termed human immunodeficiency virus type 1NC (HIV-1NC). A previously uninfected chimpanzee, C534, experienced rapid peripheral CD4+ T-cell loss to fewer than 26 cells/μl by 14 weeks after infection. CD4+ T-cell depletion was associated with high plasma HIV-1 loads but a low virus burden in the peripheral lymph node. The second chimpanzee, C459, infected 13 years previously with HIV-1LAV, experienced a more protracted course of peripheral CD4+ T-cell loss after HIV-1NCinoculation, resulting in fewer than 200 cells/μl by 96 weeks postinoculation. The quantities of viral RNA in the plasma and peripheral lymph node from C459 were below the lower limits of detection prior to inoculation with HIV-1NC but were significantly and persistently increased after superinfection, with HIV-1NC representing the predominant viral genotype. These results show that viruses derived from C499 are more pathogenic for chimpanzees than any other HIV-1 isolates described to date.

Gp120-alum boosting of a Gag-Pol-Env DNA/MVA AIDS vaccine: Poorer control of a pathogenic viral challenge

Envelope protein immunogens may improve DNA or live-vectored HIV vaccines by complementing antiviral cellular responses with Env antibodies. We tested this concept by administering two immunizations of alum-adjuvanted HIV-1 89.6 gp120 to macaques being primed at weeks 0 and 8 with SHIV 89.6 Gag-Pol-Env DNA and boosted at week 24 with SHIV-89.6 Gag-Pol-Env recombinant modified vaccinia Ankara (MVA). Three hundred micrograms of gp120 was delivered with the second DNA prime and the MVA booster. Eight months after vaccination, all animals were challenged intrarectally with the related, yet serologically distinct, SHIV-89.6P. The gp120 immunizations raised binding, but not neutralizing antibody for the challenge virus, and allowed testing of whether gp120 vaccines that fail to raise neutralizing antibody can improve protection. Following the second gp120 immunization, the plus-gp120 group showed >10 times higher levels of binding antibody than the minus-gp120 group. These levels fell and were overall similar in both groups at the time of challenge. Following the second challenge, both groups had similar temporal patterns and heights of binding and neutralizing antibodies. However, the plus-gp120 group had less consistent control of viremia and higher levels of plasma viral RNA for the first year postchallenge. Assays for complement-dependent enhancing antibody revealed a trend toward higher levels of activity in the plus-gp120 group. This trend did not reach significance in our animal groups of 8. We conclude that gp120 inoculations that fail to raise neutralizing antibody do not improve the efficacy of Gag-Pol-Env DNA/MVA vaccines.

Early Induction of Polyfunctional Simian Immunodeficiency Virus (SIV)-Specific T Lymphocytes and Rapid Disappearance of SIV from Lymph Nodes of Sooty Mangabeys during Primary Infection

Although the cellular immune response is essential for controlling SIV replication in Asian macaques, its role in maintaining nonpathogenic SIV infection in natural hosts such as sooty mangabeys (SM) remains to be defined. We have previously shown that similar to rhesus macaques (RM), SM are able to mount a T lymphocyte response against SIV infection. To investigate early control of SIV replication in natural hosts, we performed a detailed characterization of SIV-specific cellular immunity and viral control in the first 6 mo following SIV infection in SM. Detection of the initial SIV-specific IFN-γ ELISPOT response in SIVsmE041-infected SM coincided temporally with a decline in peak plasma viremia and was similar in magnitude, specificity, and breadth to SIVsmE041-infected and SIVmac239-infected RM. Despite these similarities, SM showed a greater reduction in postpeak plasma viremia and a more rapid disappearance of productively SIV-infected cells from the lymph node compared with SIVmac239-infected RM. The early Gag-specific CD8+ T lymphocyte response was significantly more polyfunctional in SM compared with RM, and granzyme B-positive CD8+ T lymphocytes were present at significantly higher frequencies in SM even prior to SIV infection. These findings suggest that the early SIV-specific T cell response may be an important determinant of lymphoid tissue viral clearance and absence of lymph node immunopathology in natural hosts of SIV infection.

Probable deceleration of progression of simian AIDS affected by opiate dependency: studies with a rhesus macaque/SIVsmm9 model.

Objectives:To determine effects of opiate dependency on development of simian AIDS. Design:Assessments of viral, immune, and clinicopathological status were conducted on rhesus macaques before and after establishment of opiate dependency and Simian Immunodeficiency Virus, sooty mangabey, strain-9 (SIVsmm9) infection. Controls received saline. Methods:Blood was collected at baseline, before opiate dependencies, and viral infections were established and then after SIVsmm9 infection, longitudinally, through 216 weeks. Plasma viral titers were assessed using the branched chain DNA assay and CD4 and CD8 counts via cytofluorometry. Clinicopathological assessments of AIDS were founded on Centers for Disease Control and Prevention and other selected criteria. Results:AIDS progression rates seemed to be decelerated and survival times increased by opiate dependency. Mean viral titers were unaffected by opiate exposure. Opiate-dependent monkeys that evidenced high initial viral titers survived significantly longer than controls. Several opiate-dependent monkeys maintained high viral titers for atypically extended durations. Several (5/19) opiate-dependent monkeys died or were removed early from the study due to “non-AIDS” causes. Conclusions:Long-term opiate dependency seemed to decelerate the rate of progression to AIDS in the SIVsmm9 monkey model. This effect was most evident in monkeys with high initial viral titers/set points. “Non-AIDS” morbidities and mortalities were noted as potential confounds of epidemiological assessments of the role of opiates in HIV/AIDS.

In vivo cell and tissue tropism of SIVsmmPBj14-bcl.3.

To gain insight into the unique pathogenicity of simian immunodeficiency virus (SIV) variant PBj14, which produces an acutely lethal enteropathic syndrome in infected pigtail macaques, we investigated the cell and tissue tropisms of a highly pathogenic biologic clone (bcl.3) of SIVsmmPBj14. To compare the relative amount of viral antigen in lymphoid organs of infected macaques we used an objective semiquantitative immunohistochemistry (sQIHC) assay. We found that in all animals viral antigen load was greater in alimentary-associated lymphoid tissues (gut-associated lymphoid tissue [GALT], tonsil, mesenteric and retropharyngeal lymph nodes) than in non-alimentary-associated lymphoid tissues (spleen, thymus, inguinal and axillary lymph nodes). Moreover, in six of nine animals examined, virus load in GALT was greater than that in any other lymphoid tissue. To determine whether the acute pathogenicity and prolific replication of SIVsmmPBj14 might be explained by a broader in vivo cell tropism than is typical of SIVs, we used cell subset separation and nested PCR. We found that the primary target cells in mesenteric lymph node for SIVsmmPBj14 were CD4+ T lymphocytes. However, the virus also infected macrophages, as well as CD8+ T cells and B cells, albeit at low frequencies. These results suggest that alimentary lymphoid tissue localization rather than unusual cell phenotype tropism distinguishes the singular pathogenesis of SIVsmmPBj14.

Reduced genetic diversity in lymphoid and central nervous system tissues and selection-induced tissue-specific compartmentalization of neuropathogenic SIVsmmFGb during acute infection.

The simian lentivirus strain SIVsmmFGb is a viral swarm population inducing neuropathology in over 90% of infected pigtailed macaques and serves as a reliable model for HIV neuropathogenesis. However, little is understood about the genetic diversity of this virus, how said diversity influences the initial seeding of the central nervous system and lymph nodes, or whether the virus forms distinct genetic compartments between tissues during acute infection. In this study, we establish that our SIVsmmFGb stock virus contains four genetically distinct envelope V1 region groups, three distinct integrase groups, and two Nef groups. We demonstrate that initial central nervous system and lymph node seeding reduces envelope V1 and integrase genetic diversity but has a variable effect on Nef diversity. SIVsmmFGb envelope V1 region genes from the basal ganglia, cerebellum, and hippocampus form distinct genetic compartments from each other, the midfrontal cortex, and the lymph nodes. Basal ganglia, cerebellum, hippocampus, and midfrontal cortex-derived nef genes all form distinct genetic compartments from each other, as well as from the lymph nodes. We also find basal ganglia, hippocampus, and midfrontal cortex-derived integrase sequences forming distinct compartments from both of the lymph nodes and that the hippocampus and midfrontal cortex form separate compartments from the cerebellum, while the axillary and mesenteric lymph nodes compartmentalize separately from each other. Compartmentalization of the envelope V1 genes resulted from positive selection, and compartmentalization of the nef and integrase genes from negative selection. These results indicate restrictions on virus genetic diversity during initial tissue seeding in neuropathogenic SIV infection.