Rama Rao Amara

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.

Broadly cross-reactive antibodies dominate the human B cell response against 2009 pandemic H1N1 influenza virus infection

Although scarce after annual influenza vaccination, B cells producing antibodies capable of neutralizing multiple influenza strains are abundant in humans infected with pandemic 2009 H1N1 influenza.

Enhancing SIV-Specific Immunity In Vivo by PD-1 Blockade

Chronic immunodeficiency virus infections are characterized by dysfunctional cellular and humoral antiviral immune responses. As such, immune modulatory therapies that enhance and/or restore the function of virus-specific immunity may protect from disease progression. Here we investigate the safety and immune restoration potential of blockade of the co-inhibitory receptor programmed death 1 (PD-1) during chronic simian immunodeficiency virus (SIV) infection in macaques. We demonstrate that PD-1 blockade using an antibody to PD-1 is well tolerated and results in rapid expansion of virus-specific CD8 T cells with improved functional quality. This enhanced T-cell immunity was seen in the blood and also in the gut, a major reservoir of SIV infection. PD-1 blockade also resulted in proliferation of memory B cells and increases in SIV envelope-specific antibody. These improved immune responses were associated with significant reductions in plasma viral load and also prolonged the survival of SIV-infected macaques. Blockade was effective during the early (week 10) as well as late (∼week 90) phases of chronic infection even under conditions of severe lymphopenia. These results demonstrate enhancement of both cellular and humoral immune responses during a pathogenic immunodeficiency virus infection by blocking a single inhibitory pathway and identify a novel therapeutic approach for control of human immunodeficiency virus infections.

Multiple-Cytokine-Producing Antiviral CD4 T Cells Are Functionally Superior to Single-Cytokine-Producing Cells

ABSTRACT Virus-specific CD4 T cells are endowed with multiple functions, such as cytokine production, CD40 ligand (CD40L) expression (associated with the costimulation of CD8 and B cells), and degranulation (associated with cytotoxic potential). Here, we used antiviral CD4 T cells present in human blood to evaluate the relationship between cytokine production and other functions of CD4 T cells. Antiviral CD4 T cells specific for a virus causing persistent infection, cytomegalovirus (CMV), and two viruses causing nonpersistent infections, influenza virus and the smallpox vaccine virus (vaccinia virus), were studied. CD4 T cells specific for each of the viruses produced all seven possible combinations of the cytokines gamma interferon (IFN-γ), interleukin-2, and tumor necrosis factor alpha. Cells producing three or two cytokines (triple producers and double producers) represented nearly 50% of the total response to each of the viruses. Triple producers expressed the highest levels of cytokines per cell, and single producers expressed the lowest. Following stimulation, higher frequencies of triple producers than single producers expressed CD40L. Only CMV-specific CD4 T cells underwent degranulation. However, higher frequencies of CMV-specific triple producers than single producers showed this functional characteristic. In contrast to the functional phenotypes, the memory phenotypes of triple producers and IFN-γ single producers did not differ. These results demonstrate a strong positive association between the cytokine coproduction capacity of a virus-specific CD4 T cell and its other functional characteristics and suggest that vaccines should aim to elicit T cells that coproduce more than one cytokine.

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.

Different Patterns of Immune Responses but Similar Control of a Simian-Human Immunodeficiency Virus 89.6P Mucosal Challenge by Modified Vaccinia Virus Ankara (MVA) and DNA/MVA Vaccines

ABSTRACT Recently we demonstrated the control of a mucosal challenge with a pathogenic chimera of simian and human immunodeficiency virus (SHIV-89.6P) by priming with a Gag-Pol-Env-expressing DNA and boosting with a Gag-Pol-Env-expressing recombinant modified vaccinia virus Ankara (DNA/MVA) vaccine. Here we evaluate the ability of the MVA component of this vaccine to serve as both a prime and a boost for an AIDS vaccine. The same immunization schedule, MVA dose, and challenge conditions were used as in the prior DNA/MVA vaccine trial. Compared to the DNA/MVA vaccine, the MVA-only vaccine raised less than 1/10 the number of vaccine-specific T cells but 10-fold-higher titers of binding antibody for Env. Postchallenge, the animals vaccinated with MVA alone increased their CD8 cell numbers to levels that were similar to those seen in DNA/MVA-vaccinated animals. However, they underwent a slower emergence and contraction of antiviral CD8 T cells and were slower to generate neutralizing antibodies than the DNA/MVA-vaccinated animals. Despite this, by 5 weeks postchallenge, the MVA-only-vaccinated animals had achieved as good control of the viral infection as the DNA/MVA group, a situation that has held up to the present time in the trial (48 weeks postchallenge). Thus, MVA vaccines, as well as DNA/MVA vaccines, merit further evaluation for their ability to control the current AIDS pandemic.

Elevated expression levels of inhibitory receptor programmed death 1 on simian immunodeficiency virus-specific CD8 T cells during chronic infection but not after vaccination.

ABSTRACT Here, we study the temporal expression of the inhibitory receptor programmed death 1 (PD-1) on simian immunodeficiency virus (SIV) Gag-specific T cells following pathogenic SIV infection or following vaccination with a DNA/modified vaccinia virus Ankara (DNA/MVA) vaccine and simian/human immunodeficiency virus (SHIV) challenge in macaques. Following infection, the majority (>95%) of Gag-specific CD8 T cells expressed PD-1, and the level of PD-1 expression per cell increased over time. The level of PD-1 expression in lymph nodes and rectal mucosal tissue, the major sites of virus replication, was higher compared to blood. In vitro blockade of PD-1 resulted in enhanced proliferation of SIV-specific CD8 as well as CD4 T cells. In contrast, following vaccination, the majority of peak effector Gag-specific CD8 T cells expressed low levels of PD-1, and these levels decreased further as the cells differentiated into memory cells. In addition, following SHIV challenge of these vaccinated macaques, the level of PD-1 expression on Gag-specific CD8 T cells correlated positively with plasma viremia. These results demonstrate that SIV-specific CD8 T cells express PD-1 after exposure to antigen but downregulate expression under conditions of antigen clearance and enhance expression under conditions of antigen persistence. They also demonstrate that the level of PD-1 expression per cell rather than the presence or absence of expression plays an important role in regulating CD8 T-cell dysfunction in pathogenic SIV infection. In addition, they demonstrate that similar to HIV infection, the PD-1:PD-1 ligand inhibitory pathway is operational in pathogenic SIV infection, and the macaque/SIV model would be ideal to test the safety and therapeutic benefit of blocking this pathway in vivo.

Human Immunodeficiency Virus Type 1 Controllers but Not Noncontrollers Maintain CD4 T Cells Coexpressing Three Cytokines

ABSTRACT Here, we evaluate the cytokine coexpression profiles of human immunodeficiency virus (HIV)-specific CD4 T cells for the expression of the cytokines gamma interferon (IFN-γ), interleukin-2, and tumor necrosis factor alpha. In controllers, CD4 T cells producing three or two cytokines (triple producers and double producers, respectively) represented >50% of the total response. In contrast, in noncontrollers ∼75% of responding cells produced only one cytokine (single producers), mostly IFN-γ. Cells producing three cytokines were functionally superior to those producing single cytokines and showed an inverse correlation (P < 0.001) with viral load. These results demonstrate a strong association between the maintenance of highly functional CD4 T cells producing three cytokines and control of HIV-1.

Apoptosis-mediated enhancement of DNA-raised immune responses by mutant caspases.

Apoptotic bodies can be used to target delivery of DNA-expressed immunogens into professional antigen-presenting cells (APCs). Here we show that antigen-laden apoptotic bodies created by vectors co-expressing influenza virus hemagglutinin (HA) or nucleoprotein (NP) genes and mutant caspase genes markedly increased T-cell responses. Both CD8 and CD4 T-cell responses were affected. The adjuvant activity was restricted to partially inactivated caspases that allowed immunogen expression before the generation of apoptotic bodies. Active-site mutants of murine caspase 2 and an autocatalytic chimera of murine caspase 2 prodomain and human caspase 3 induced apoptosis that did not interfere with immunogen expression. The adjuvant activity also enhanced B-cell responses, but to a lesser extent than T-cell responses. The large increases in T-cell responses represent one of the strongest effects to date of a DNA adjuvant on cellular immunity.

Long-Lived Poxvirus Immunity, Robust CD4 Help, and Better Persistence of CD4 than CD8 T Cells

ABSTRACT The currently used smallpox vaccine is associated with a high incidence of adverse events, and there is a serious need for a safe and effective alternative vaccine. Here, we carried out a longitudinal evaluation of vaccinia virus-specific CD4 and CD8 T cells in smallpox-vaccinated individuals by using a highly sensitive intracellular cytokine staining assay. Our results demonstrate that, in addition to the CD8 response, the smallpox vaccinations raised a robust CD4 response with a Th1-dominant cytokine profile. These CD4 T cells were stable and exhibited only a twofold contraction between peak effector and memory phases compared with an approximate sevenfold contraction for CD8 cells. A significant proportion of vaccinated individuals lost detectable CD8 memory while maintaining CD4 memory. After a booster immunization, these individuals generated a robust CD8 response, which some of them rapidly lost. Thus, the current smallpox vaccine provides long-lasting CD4 help that may be critical for long-lived B-cell memory. We suggest that the provision of adequate CD4 help for CD8 and humoral effector functions will be critical to the success of the next generation of smallpox vaccines.