Jeffrey E. Teigler

Vaccination with Adenovirus Serotypes 35, 26, and 48 Elicits Higher Levels of Innate Cytokine Responses than Adenovirus Serotype 5 in Rhesus Monkeys

ABSTRACT Adenovirus (Ad) vaccine vectors have proven highly immunogenic in multiple experimental models, but the innate immune responses induced by these vectors remain poorly characterized. Here we report innate cytokine responses to 5 different Ad vectors in 26 rhesus monkeys. Vaccination with adenovirus serotype 35 (Ad35), Ad26, and Ad48 induced substantially higher levels of antiviral (gamma interferon [IFN-γ], 10-kDa gamma interferon-induced protein [IP-10]) and proinflammatory (interleukin 1 receptor antagonist [IL-1RA], IL-6) cytokines than vaccination with Ad5 on day 1 following immunization. In vitro studies with capsid chimeric vectors and receptor-blocking monoclonal antibodies suggested that fiber-receptor interactions, as well as other capsid components, were critical for triggering these innate responses. Moreover, multiple cell populations, including dendritic cells, monocytes/macrophages, and T lymphocytes, contributed to these innate cytokine profiles. These data demonstrate that Ad35, Ad26, and Ad48, which utilize CD46 as their primary cellular receptor, induce significantly greater innate cytokine responses than Ad5, which uses the coxsackievirus and adenovirus receptor (CAR). These differences in innate triggering result in markedly different immunologic milieus for the subsequent generation of adaptive immune responses by these vaccine vectors.

Identification of a Novel C-Terminal Cleavage of Crimean-Congo Hemorrhagic Fever Virus PreGN That Leads to Generation of an NSM Protein

ABSTRACT The structural glycoproteins of Crimean-Congo hemorrhagic fever virus (CCHFV; genus Nairovirus, family Bunyaviridae) are derived through endoproteolytic cleavage of a 1,684-amino-acid M RNA segment-encoded polyprotein. This polyprotein is cotranslationally cleaved into the PreGN and PreGC precursors, which are then cleaved by SKI-1 and a SKI-1-like protease to generate the N termini of GN and GC, respectively. However, the resulting polypeptide defined by the N termini of GN and GC is predicted to be larger (58 kDa) than mature GN (37 kDa). By analogy to the topologically similar M segment-encoded polyproteins of viruses in the Orthobunyavirus genus, the C-terminal region of PreGN that contains four predicted transmembrane domains may also contain a nonstructural protein, NSM. To characterize potential PreGN C-terminal cleavage events, a panel of epitope-tagged PreGN truncation and internal deletion mutants was developed. These constructs allowed for the identification of a C-terminal endoproteolytic cleavage within, or very proximal to, the second predicted transmembrane domain following the GN ectodomain and the subsequent generation of a C-terminal fragment. Pulse-chase experiments showed that PreGN C-terminal cleavage occurred shortly after synthesis of the precursor and prior to generation of the GN glycoprotein. The resulting fragment trafficked to the Golgi compartment, the site of virus assembly. Development of an antiserum specific to the second cytoplasmic loop of PreGN allowed detection of cell-associated NSM proteins derived from transient expression of the complete CCHFV M segment and also in the context of virus infection.

The Canarypox Virus Vector ALVAC Induces Distinct Cytokine Responses Compared to the Vaccinia Virus-Based Vectors MVA and NYVAC in Rhesus Monkeys

ABSTRACT Despite the growing use of poxvirus vectors as vaccine candidates for multiple pathogens and cancers, their innate stimulatory properties remain poorly characterized. Here we show that the canarypox virus-based vector ALVAC induced distinct systemic proinflammatory and antiviral cytokine and chemokine levels following the vaccination of rhesus monkeys compared to the vaccinia virus-based vectors MVA and NYVAC. These data suggest that there are substantial biological differences among leading poxvirus vaccine vectors that may influence resultant adaptive immune responses following vaccination.

Vaccine-elicited CD4 T cells induce immunopathology after chronic LCMV infection.

For vaccines, CD4+ T cells can spell trouble The ideal vaccine elicits immune memory—either antibodies or memory T cells—to protect the host from subsequent infections. T cell–mediated immunity requires both helper CD4+ T cells and cytotoxic CD8+ T cells to kill virus-infected cells. But what happens when a vaccine only elicits CD4+ memory T cells? Penaloza-MacMaster et al. probed this question by giving mice a vaccine that generated only memory CD4+ T cells against lymphocytic choriomeningitis virus (LCMV). Instead of protecting mice against chronic LCMV, vaccinated mice developed massive inflammation and died. Virus-specific CD8+ T cells or antibodies protected mice from the pathology. These results may have implications for vaccines against chronic viruses such as HIV. Science, this issue p. 278 Severe immunopathology kills virally infected mice that received vaccines targeting only CD4+ T cells. CD4 T cells promote innate and adaptive immune responses, but how vaccine-elicited CD4 T cells contribute to immune protection remains unclear. We evaluated whether induction of virus-specific CD4 T cells by vaccination would protect mice against infection with chronic lymphocytic choriomeningitis virus (LCMV). Immunization with vaccines that selectively induced CD4 T cell responses resulted in catastrophic inflammation and mortality after challenge with a persistent strain of LCMV. Immunopathology required antigen-specific CD4 T cells and was associated with a cytokine storm, generalized inflammation, and multi-organ system failure. Virus-specific CD8 T cells or antibodies abrogated the pathology. These data demonstrate that vaccine-elicited CD4 T cells in the absence of effective antiviral immune responses can trigger lethal immunopathology.

Construction and Evaluation of Novel Rhesus Monkey Adenovirus Vaccine Vectors

ABSTRACT Adenovirus vectors are widely used as vaccine candidates for a variety of pathogens, including HIV-1. To date, human and chimpanzee adenoviruses have been explored in detail as vaccine vectors. The phylogeny of human and chimpanzee adenoviruses is overlapping, and preexisting humoral and cellular immunity to both are exhibited in human populations worldwide. More distantly related adenoviruses may therefore offer advantages as vaccine vectors. Here we describe the primary isolation and vectorization of three novel adenoviruses from rhesus monkeys. The seroprevalence of these novel rhesus monkey adenovirus vectors was extremely low in sub-Saharan Africa human populations, and these vectors proved to have immunogenicity comparable to that of human and chimpanzee adenovirus vaccine vectors in mice. These rhesus monkey adenoviruses phylogenetically clustered with the poorly described adenovirus species G and robustly stimulated innate immune responses. These novel adenoviruses represent a new class of candidate vaccine vectors. IMPORTANCE Although there have been substantial efforts in the development of vaccine vectors from human and chimpanzee adenoviruses, far less is known about rhesus monkey adenoviruses. In this report, we describe the isolation and vectorization of three novel rhesus monkey adenoviruses. These vectors exhibit virologic and immunologic characteristics that make them attractive as potential candidate vaccine vectors for both HIV-1 and other pathogens.

Late Endosomal Trafficking of Alternative Serotype Adenovirus Vaccine Vectors Augments Antiviral Innate Immunity

ABSTRACT Adenovirus (Ad) vaccine vectors have found widespread use as vaccine platforms against multiple infections and cancers, and multiple serotypes have been shown to differ significantly in their biological properties and immune phenotypes. Our laboratory and others have previously described differential innate immune stimulation elicited by various Ad serotypes. Here, we show that Ad serotype 5 (Ad5) traffics rapidly to the nucleus following infection, whereas Ad35 and Ad26 accumulate in late endosomes 2 to 8 h postinfection. Innate immune cytokine elicitation by all Ad serotypes was abrogated by blockade of endosomal acidification, cathepsin B, and caspase 1, suggesting that virus interactions with acid-dependent sensors, such as Toll-like receptor- and cathepsin-dependent inflammasome activation in late endosomes, may trigger innate immunity. These data suggest a mechanism by which Ad vectors from various serotypes differentially trigger innate antiviral pathways via distinct intracellular trafficking to late endosomes. IMPORTANCE Adenoviruses (Ad) are widely used for vaccination and gene therapy applications. Importantly, Ad vectors have been shown to differ significantly in their innate immune profiles both in vivo and in vitro. The molecular mechanism that underlies these observed differences has important implications for the development of improved vaccines. In this study, we propose a mechanism in which the degree of late endosomal trafficking of Ad vectors results in differential stimulation of late endosomal pattern recognition receptors.

Hexon Hypervariable Region-Modified Adenovirus Type 5 (Ad5) Vectors Display Reduced Hepatotoxicity but Induce T Lymphocyte Phenotypes Similar to Ad5 Vectors

ABSTRACT Hexon modification of adenovirus type 5 (Ad5) vectors with the hypervariable regions (HVRs) of Ad48 has been shown to allow Ad5HVR48 vectors to circumvent the majority of the preexisting Ad5-neutralizing antibodies. However, it remains unclear whether modifying hexon HVRs impacts innate or adaptive immune responses elicited by this vector. In this study, we investigated the influence of the HVR substitution of Ad5 on innate and adaptive immune responses following vaccination. Ad5HVR48 displayed an intermediate level of innate immune cytokines and chemokines relative to those of Ad5 and Ad48, consistent with its chimeric nature. Hepatotoxicity was observed after Ad5 immunization but not after Ad5HVR48 or Ad48 immunization. However, the CD8+ T-cell responses elicited by Ad5HVR48 vectors displayed a partially exhausted phenotype, as evidenced by the sustained expression of programmed death 1 (PD-1), decreased effector-to-central memory conversion, and reduced memory recall responses, similar to those elicited by Ad5 vectors and in contrast to those induced by Ad48 vectors. Taken together, these results indicate that although Ad5HVR48 largely bypasses preexisting Ad5 neutralizing antibodies and shows reduced hepatotoxicity compared to that of Ad5, it induces adaptive immune phenotypes that are functionally exhausted similar to those elicited by Ad5.

Augmented Replicative Capacity of the Boosting Antigen Improves the Protective Efficacy of Heterologous Prime-Boost Vaccine Regimens

ABSTRACT Prime-boost immunization regimens have proven efficacious at generating robust immune responses. However, whether the level of replication of the boosting antigen impacts the magnitude and protective efficacy of vaccine-elicited immune responses remains unclear. To evaluate this, we primed mice with replication-defective adenovirus vectors expressing the lymphocytic choriomeningitis virus (LCMV) glycoprotein (GP), followed by boosting with either LCMV Armstrong, which is rapidly controlled, or LCMV CL-13, which leads to a more prolonged exposure to the boosting antigen. Although priming of naive mice with LCMV CL-13 normally results in T cell exhaustion and establishment of chronic infection, boosting with CL-13 resulted in potent recall CD8 T cell responses that were greater than those following boosting with LCMV Armstrong. Furthermore, following the CL-13 boost, a greater number of anamnestic CD8 T cells localized to the lymph nodes, exhibited granzyme B expression, and conferred improved protection against Listeria and vaccinia virus challenges compared with the Armstrong boost. Overall, our findings suggest that the replicative capacity of the boosting antigen influences the protective efficacy afforded by prime-boost vaccine regimens. These findings are relevant for optimizing vaccine candidates and suggest a benefit of robustly replicating vaccine vectors. IMPORTANCE The development of optimal prime-boost vaccine regimens is a high priority for the vaccine development field. In this study, we compared two boosting antigens with different replicative capacities. Boosting with a more highly replicative vector resulted in augmented immune responses and improved protective efficacy.

Distinct biomarker signatures in HIV acute infection associate with viral dynamics and reservoir size

Estimating the size of the viral reservoir is critical for HIV cure strategies. Biomarkers in peripheral circulation may give insights into the establishment of the viral reservoir in compartments not easily accessible. We therefore measured systemic levels of 84 soluble biomarkers belonging to a broad array of immune pathways in acute HIV infection in both antiretroviral therapy-naive (ART-naive) individuals as well as individuals who began ART upon early detection of HIV infection. These biomarkers were measured longitudinally during acute and chronic infection and their relationship to viral reservoir establishment and persistence was assessed. We observed several distinct biomarker pathways induced following HIV infection such as IFN-γ-signaled chemokines, proinflammatory markers, and TNF-α-family members. Levels of several of these factors directly correlated with contemporaneous viral loads and/or frequency of peripheral blood mononuclear cells harboring HIV DNA during acute HIV infection. MCP-1, MIP-3β, sTNFR-II, and IL-10 levels prior to ART associated with HIV DNA levels after 96 weeks of treatment, suggesting a link between early immune signaling events and the establishment and persistence of the viral reservoir during ART. Furthermore, they offer potentially novel tools for gaining insight into relative reservoir size in acutely infected individuals and the potential of associated risks of treatment interruption.

Adenovirus vectors from various serotypes induce distinct cytokine profiles

Results Ad35 and Ad26 induced higher levels of antiviral and proinflammatory cytokines (e.g. IFNa2, IFNg, IL-1b) compared to Ad5 in human PBMC (p<0.01, KruskalWallis test; Dunn’s correction). Replacement of Ad5 fiber with that of Ad35 (Ad5f35) increased cytokine induction, while Ad35f5 displayed decreased stimulation, indicating the importance of fiber-receptor interactions for innate immune stimulation. Similarly, monkeys vaccinated with Ad35 or Ad26 also displayed markedly higher levels of antiviral and proinflammatory cytokines compared to Ad5 on day 1 post-vaccination (p<0.05, Mann-Whitney U test).