Srinivas S. Rao

Microbial translocation is a cause of systemic immune activation in chronic HIV infection

Chronic activation of the immune system is a hallmark of progressive HIV infection and better predicts disease outcome than plasma viral load, yet its etiology remains obscure. Here we show that circulating microbial products, probably derived from the gastrointestinal tract, are a cause of HIV-related systemic immune activation. Circulating lipopolysaccharide, which we used as an indicator of microbial translocation, was significantly increased in chronically HIV-infected individuals and in simian immunodeficiency virus (SIV)-infected rhesus macaques (P ≤ 0.002). We show that increased lipopolysaccharide is bioactive in vivo and correlates with measures of innate and adaptive immune activation. Effective antiretroviral therapy seemed to reduce microbial translocation partially. Furthermore, in nonpathogenic SIV infection of sooty mangabeys, microbial translocation did not seem to occur. These data establish a mechanism for chronic immune activation in the context of a compromised gastrointestinal mucosal surface and provide new directions for therapeutic interventions that modify the consequences of acute HIV infection.

Preserved CD4+ Central Memory T Cells and Survival in Vaccinated SIV-Challenged Monkeys

Vaccine-induced cellular immunity controls virus replication in simian immunodeficiency virus (SIV)–infected monkeys only transiently, leading to the question of whether such vaccines for AIDS will be effective. We immunized monkeys with plasmid DNA and replication-defective adenoviral vectors encoding SIV proteins and then challenged them with pathogenic SIV. Although these monkeys demonstrated a reduction in viremia restricted to the early phase of SIV infection, they showed a prolonged survival. This survival was associated with preserved central memory CD4+ T lymphocytes and could be predicted by the magnitude of the vaccine-induced cellular immune response. These immune correlates of vaccine efficacy should guide the evaluation of AIDS vaccines in humans.

Induction of Broadly Neutralizing H1N1 Influenza Antibodies by Vaccination

Toward a General Flu Vaccination Current seasonal influenza virus vaccines are targeted against specific viral strains and do not provide broad, durable protection. Seasonal influenza vaccines induce protective antibody responses against regions of viral hemagglutinin (HA) that rapidly mutate so that very soon, the virus becomes resistant to vaccination. Conserved regions of HA also exist, and a major goal of influenza vaccine development is to design a vaccine that elicits antibodies against the conserved regions so that protection against a wide range of viral strains is achieved. Wei et al. (p. 1060, published online 15 July; see the Perspective by Doms) show that a combined HA DNA prime, followed by boosting with a seasonal vaccine, elicits broadly cross-reactive neutralizing antibody responses in mice, ferrets, and nonhuman primates, which were protective in mice and ferrets against heterologous influenza challenge. The neutralizing antibodies were directed against the conserved HA stem region, which indicates the possibility that a more broadly protective vaccine against influenza could be developed. An influenza virus vaccine elicits a broadly neutralizing, cross-protective antibody response in mice, ferrets, and nonhuman primates. The rapid dissemination of the 2009 pandemic influenza virus underscores the need for universal influenza vaccines that elicit protective immunity to diverse viral strains. Here, we show that vaccination with plasmid DNA encoding H1N1 influenza hemagglutinin (HA) and boosting with seasonal vaccine or replication-defective adenovirus 5 vector encoding HA stimulated the production of broadly neutralizing influenza antibodies. This prime/boost combination increased the neutralization of diverse H1N1 strains dating from 1934 to 2007 as compared to either component alone and conferred protection against divergent H1N1 viruses in mice and ferrets. These antibodies were directed to the conserved stem region of HA and were also elicited in nonhuman primates. Cross-neutralization of H1N1 subtypes elicited by this approach provides a basis for the development of a universal influenza vaccine for humans.

Structure-based design of a fusion glycoprotein vaccine for respiratory syncytial virus.

Designer Vaccine Respiratory syncytial virus (RSV) is one of the last remaining childhood diseases without an approved vaccine. Using a structure-based approach, McLellan et al. (p. 592) designed over 150 fusion glycoprotein variants, assessed their antibody reactivity, determined crystal structures of stabilized variants, and measured their ability to elicit protective responses. This approach yielded an immunogen that elicits higher protective responses than the postfusion form of the fusion glycoprotein, which is one of the current leading RSV vaccine candidates entering clinical trials. Importantly, highly protective responses were elicited in both mice and macaques. Molecular engineering of a childhood virus surface protein significantly improves protective responses in mice and macaques. Respiratory syncytial virus (RSV) is the leading cause of hospitalization for children under 5 years of age. We sought to engineer a viral antigen that provides greater protection than currently available vaccines and focused on antigenic site Ø, a metastable site specific to the prefusion state of the RSV fusion (F) glycoprotein, as this site is targeted by extremely potent RSV-neutralizing antibodies. Structure-based design yielded stabilized versions of RSV F that maintained antigenic site Ø when exposed to extremes of pH, osmolality, and temperature. Six RSV F crystal structures provided atomic-level data on how introduced cysteine residues and filled hydrophobic cavities improved stability. Immunization with site Ø–stabilized variants of RSV F in mice and macaques elicited levels of RSV-specific neutralizing activity many times the protective threshold.

A virus-like particle vaccine for epidemic Chikungunya virus protects nonhuman primates against infection

Chikungunya virus (CHIKV) has infected millions of people in Africa, Europe and Asia since this alphavirus reemerged from Kenya in 2004. The severity of the disease and the spread of this epidemic virus present a serious public health threat in the absence of vaccines or antiviral therapies. Here, we describe a new vaccine that protects against CHIKV infection of nonhuman primates. We show that selective expression of viral structural proteins gives rise to virus-like particles (VLPs) in vitro that resemble replication-competent alphaviruses. Immunization with these VLPs elicited neutralizing antibodies against envelope proteins from alternative CHIKV strains. Monkeys immunized with VLPs produced high-titer neutralizing antibodies that protected against viremia after high-dose challenge. We transferred these antibodies into immunodeficient mice, where they protected against subsequent lethal CHIKV challenge, indicating a humoral mechanism of protection. Immunization with alphavirus VLP vaccines represents a strategy to contain the spread of CHIKV and related pathogenic viruses in humans.

CD4 T follicular helper cell dynamics during SIV infection.

CD4 T follicular helper (TFH) cells interact with and stimulate the generation of antigen-specific B cells. TFH cell interaction with B cells correlates with production of SIV-specific immunoglobulins. However, the fate of TFH cells and their participation in SIV-induced antibody production is not well understood. We investigated the phenotype, function, location, and molecular signature of TFH cells in rhesus macaques. Similar to their human counterparts, TFH cells in rhesus macaques represented a heterogeneous population with respect to cytokine function. In a highly differentiated subpopulation of TFH cells, characterized by CD150lo expression, production of Th1 cytokines was compromised while IL-4 production was augmented, and cells exhibited decreased survival, cycling, and trafficking capacity. TFH cells exhibited a distinct gene profile that was markedly altered by SIV infection. TFH cells were infected by SIV; yet, in some animals, these cells actually accumulated during chronic SIV infection. Generalized immune activation and increased IL-6 production helped drive TFH differentiation during SIV infection. Accumulation of TFH cells was associated with increased frequency of activated germinal center B cells and SIV-specific antibodies. Therefore, chronic SIV does not disturb the ability of TFH cells to help B cell maturation and production of SIV-specific immunoglobulins.

Self-assembling influenza nanoparticle vaccines elicit broadly neutralizing H1N1 antibodies.

Influenza viruses pose a significant threat to the public and are a burden on global health systems. Each year, influenza vaccines must be rapidly produced to match circulating viruses, a process constrained by dated technology and vulnerable to unexpected strains emerging from humans and animal reservoirs. Here we use knowledge of protein structure to design self-assembling nanoparticles that elicit broader and more potent immunity than traditional influenza vaccines. The viral haemagglutinin was genetically fused to ferritin, a protein that naturally forms nanoparticles composed of 24 identical polypeptides. Haemagglutinin was inserted at the interface of adjacent subunits so that it spontaneously assembled and generated eight trimeric viral spikes on its surface. Immunization with this influenza nanoparticle vaccine elicited haemagglutination inhibition antibody titres more than tenfold higher than those from the licensed inactivated vaccine. Furthermore, it elicited neutralizing antibodies to two highly conserved vulnerable haemagglutinin structures that are targets of universal vaccines: the stem and the receptor binding site on the head. Antibodies elicited by a 1999 haemagglutinin–nanoparticle vaccine neutralized H1N1 viruses from 1934 to 2007 and protected ferrets from an unmatched 2007 H1N1 virus challenge. This structure-based, self-assembling synthetic nanoparticle vaccine improves the potency and breadth of influenza virus immunity, and it provides a foundation for building broader vaccine protection against emerging influenza viruses and other pathogens.

Type I interferon responses in rhesus macaques prevent SIV infection and slow disease progression

Inflammation in HIV infection is predictive of non-AIDS morbidity and death, higher set point plasma virus load and virus acquisition; thus, therapeutic agents are in development to reduce its causes and consequences. However, inflammation may simultaneously confer both detrimental and beneficial effects. This dichotomy is particularly applicable to type I interferons (IFN-I) which, while contributing to innate control of infection, also provide target cells for the virus during acute infection, impair CD4 T-cell recovery, and are associated with disease progression. Here we manipulated IFN-I signalling in rhesus macaques (Macaca mulatta) during simian immunodeficiency virus (SIV) transmission and acute infection with two complementary in vivo interventions. We show that blockade of the IFN-I receptor caused reduced antiviral gene expression, increased SIV reservoir size and accelerated CD4 T-cell depletion with progression to AIDS despite decreased T-cell activation. In contrast, IFN-α2a administration initially upregulated expression of antiviral genes and prevented systemic infection. However, continued IFN-α2a treatment induced IFN-I desensitization and decreased antiviral gene expression, enabling infection with increased SIV reservoir size and accelerated CD4 T-cell loss. Thus, the timing of IFN-induced innate responses in acute SIV infection profoundly affects overall disease course and outweighs the detrimental consequences of increased immune activation. Yet, the clinical consequences of manipulation of IFN signalling are difficult to predict in vivo and therapeutic interventions in human studies should be approached with caution.

Hemagglutinin-stem nanoparticles generate heterosubtypic influenza protection

The antibody response to influenza is primarily focused on the head region of the hemagglutinin (HA) glycoprotein, which in turn undergoes antigenic drift, thus necessitating annual updates of influenza vaccines. In contrast, the immunogenically subdominant stem region of HA is highly conserved and recognized by antibodies capable of binding multiple HA subtypes. Here we report the structure-based development of an H1 HA stem–only immunogen that confers heterosubtypic protection in mice and ferrets. Six iterative cycles of structure-based design (Gen1–Gen6) yielded successive H1 HA stabilized-stem (HA–SS) immunogens that lack the immunodominant head domain. Antigenic characterization, determination of two HA–SS crystal structures in complex with stem-specific monoclonal antibodies and cryo-electron microscopy analysis of HA–SS on ferritin nanoparticles (H1–SS–np) confirmed the preservation of key structural elements. Vaccination of mice and ferrets with H1–SS–np elicited broadly cross-reactive antibodies that completely protected mice and partially protected ferrets against lethal heterosubtypic H5N1 influenza virus challenge despite the absence of detectable H5N1 neutralizing activity in vitro. Passive transfer of immunoglobulin from H1–SS–np–immunized mice to naive mice conferred protection against H5N1 challenge, indicating that vaccine-elicited HA stem–specific antibodies can protect against diverse group 1 influenza strains.

Immune and Genetic Correlates of Vaccine Protection Against Mucosal Infection by SIV in Monkeys.

A vaccine protecting monkeys against mucosal infection by simian immunodeficiency virus sheds light on immune and genetic correlates of protection. Unraveling Immune Correlates of Vaccine Protection Developing an effective vaccine against HIV-1, the virus that causes AIDS, has been a huge challenge that has stymied AIDS researchers for several decades. A key problem for HIV vaccine trials has been the lack of immune correlates that indicate which antibody and T cell responses in the vaccinees correlate directly with a protective effect. The only HIV vaccine trial to date that has shown a protective effect is the RV144 trial carried out in Thailand between 2003 and 2006, with the final results reported in 2009. In this trial of 16,400 Thai volunteers, those vaccinated with a prime-boost HIV vaccine showed a reduction in the rate of infection by HIV-1 of 31% compared to volunteers given a placebo. The protective effect was seen for up to 3 years after the initial vaccination, but the immune correlates of protection by this vaccine are still not known. In an effort to learn more about possible immune correlates of HIV vaccine protection, Letvin and colleagues used a prime/boost vaccine regimen in monkeys that was similar to that used in the RV144 trial. Monkeys were vaccinated with a plasmid DNA prime/recombinant adenovirus serotype 5 (rAd5) boost vaccine regimen and then were challenged with intrarectal doses of one of two isolates of the simian immunodeficiency virus (SIV) every week for 12 weeks. Although the vaccine had no impact on acquisition of the SIVmac251 isolate (which is tough for the monkey immune system to neutralize), the vaccine provided a 50% reduction in infection with the SIVsmE660 isolate (which more readily undergoes neutralization). The authors then examined a variety of immune responses in the protected vaccinated monkeys including cellular, antibody, and innate immune responses; they also examined whether protective host alleles were present in the protected animals. They found that low levels of neutralizing antibodies and a CD4+ T cell response against the HIV envelope (Env) protein correlated with the protective effect. In addition, monkeys that expressed two TRIM5 alleles that help to restrict SIV replication in host cells were protected by the vaccine, whereas monkeys expressing one TRIM5 allele that is permissive for SIV replication were not. This study begins to unravel the immune and genetic correlates of protection in nonhuman primates and highlights the need to scrutinize these types of correlates in future trials of HIV vaccines in human volunteers. The RV144 vaccine trial in Thailand demonstrated that an HIV vaccine could prevent infection in humans and highlights the importance of understanding protective immunity against HIV. We used a nonhuman primate model to define immune and genetic mechanisms of protection against mucosal infection by the simian immunodeficiency virus (SIV). A plasmid DNA prime/recombinant adenovirus serotype 5 (rAd5) boost vaccine regimen was evaluated for its ability to protect monkeys from infection by SIVmac251 or SIVsmE660 isolates after repeat intrarectal challenges. Although this prime-boost vaccine regimen failed to protect against SIVmac251 infection, 50% of vaccinated monkeys were protected from infection with SIVsmE660. Among SIVsmE660-infected animals, there was about a one-log reduction in peak plasma virus RNA in monkeys expressing the major histocompatibility complex class I allele Mamu-A*01, implicating cytotoxic T lymphocytes in the control of SIV replication once infection is established. Among Mamu-A*01–negative monkeys challenged with SIVsmE660, no CD8+ T cell response or innate immune response was associated with protection against virus acquisition. However, low levels of neutralizing antibodies and an envelope-specific CD4+ T cell response were associated with vaccine protection in these monkeys. Moreover, monkeys that expressed two TRIM5 alleles that restrict SIV replication were more likely to be protected from infection than monkeys that expressed at least one permissive TRIM5 allele. This study begins to elucidate the mechanisms of vaccine protection against immunodeficiency viruses and highlights the need to analyze these immune and genetic correlates of protection in future trials of HIV vaccine strategies.