Franklin R. Toapanta

Cross-Clade Protective Immune Responses to Influenza Viruses with H5N1 HA and NA Elicited by an Influenza Virus-Like Particle

Background Vaccination is a cost-effective counter-measure to the threat of seasonal or pandemic outbreaks of influenza. To address the need for improved influenza vaccines and alternatives to egg-based manufacturing, we have engineered an influenza virus-like particle (VLP) as a new generation of non-egg or non-mammalian cell culture-based candidate vaccine. Methodology/Principal Findings We generated from a baculovirus expression system using insect cells, a non-infectious recombinant VLP vaccine from both influenza A H5N1 clade 1 and clade 2 isolates with pandemic potential. VLPs were administered to mice in either a one-dose or two-dose regimen and the immune responses were compared to those induced by recombinant hemagglutinin (rHA). Both humoral and cellular responses were analyzed. Mice vaccinated with VLPs were protected against challenge with lethal reassortant viruses expressing the H5N1 HA and NA, regardless if the H5N1 clade was homologous or heterologous to the vaccine. However, rHA-vaccinated mice showed considerable weight loss and death following challenge with the heterovariant clade virus. Protection against death induced by VLPs was independent of the pre-challenge HAI titer or cell-mediated responses to HA or M1 since vaccinated mice, with low to undetectable cross-clade HAI antibodies or cellular responses to influenza antigens, were still protected from a lethal viral challenge. However, an apparent association rate of antibody binding to HA correlated with protection and was enhanced using VLPs, particularly when delivered intranasally, compared to rHA vaccines. Conclusion/Significance This is the first report describing the use of an H5N1 VLP vaccine created from a clade 2 isolate. The results show that a non-replicating virus-like particle is effective at eliciting a broadened, cross-clade protective immune response to proteins from emerging H5N1 influenza isolates giving rise to a potential pandemic influenza vaccine candidate for humans that can be stockpiled for use in the event of an outbreak of H5N1 influenza.

Impaired immune responses in the lungs of aged mice following influenza infection

BackgroundEach year, influenza virus infection causes severe morbidity and mortality, particularly in the most susceptible groups including children, the elderly (>65 years-old) and people with chronic respiratory diseases. Among the several factors that contribute to the increased susceptibility in elderly populations are the higher prevalence of chronic diseases (e.g. diabetes) and the senescence of the immune system.MethodsIn this study, aged and adult mice were infected with sublethal doses of influenza virus (A/Puerto Rico/8/1934). Differences in weight loss, morbidity, virus titer and the kinetics of lung infiltration with cells of the innate and adaptive immune responses were analyzed. Additionally, the main cytokines and chemokines produced by these cells were also assayed.ResultsCompared to adult mice, aged mice had higher morbidity, lost weight more rapidly, and recovered more slowly from infection. There was a delay in the accumulation of granulocytic cells and conventional dendritic cells (cDCs), but not macrophages in the lungs of aged mice compared to adult animals. The delayed infiltration kinetics of APCs in aged animals correlated with alteration in their activation (CD40 expression), which also correlated with a delayed detection of cytokines and chemokines in lung homogenates. This was associated with retarded lung infiltration by natural killer (NK), CD4+ and CD8+ T-cells. Furthermore, the percentage of activated (CD69+) influenza-specific and IL-2 producer CD8+ T-cells was higher in adult mice compared to aged ones. Additionally, activation (CD69+) of adult B-cells was earlier and correlated with a quicker development of neutralizing antibodies in adult animals.ConclusionOverall, alterations in APC priming and activation lead to delayed production of cytokines and chemokines in the lungs that ultimately affected the infiltration of immune cells following influenza infection. This resulted in delayed activation of the adaptive immune response and subsequent delay in clearance of virus and prolonged illness in aged animals. Since the elderly are the fastest growing segment of the population in developed countries, a better understanding of the changes that occur in the immune system during the aging process is a priority for the development of new vaccines and adjuvants to improve the immune responses in this population.

Complement-mediated activation of the adaptive immune responses: role of C3d in linking the innate and adaptive immunity.

C3d is the final degradation product of the third component of complement (C3). When conjugated to an antigen, C3d enhances immune responses to the fused antigen. Therefore, this molecule has been used as an adjuvant to enhance the immune responses to various foreign and self-proteins. C3d binds to the complement receptor 2 (CR2) that is located on the surface of follicular dendritic cells (FDC), B cells, and T cells in many species. C3d stimulates antigen presentation by FCD and helps to maintain immunological B cell memory. On B cells, C3d interaction with CR2 will collect molecules such as CD19, TAPA (CD81), and Lew 13. CD19 has a long intracellular tail that triggers a signaling cascade that results in cell activation and proliferation. Furthermore, simultaneous C3d-CR2 ligation and surface immunoglobulin (sIg) by antigen, activates two signaling pathways that cross-talk and synergize to activate B cells. However, C3d can enhance antibody titers in the absence of CR2 binding, indicating CR2-independent mechanism(s) of enhancement of the immune response. This review focuses on the complexity of the C3d-CR2 interaction, the importance of this interaction for the enhancement of the immune response by C3d and its derivatives, as well as the paradoxical enhancement of the immune response in the absence of CR2.

Cutting Edge: C3d Functions as a Molecular Adjuvant in the Absence of CD21/35 Expression

Complement component C3 covalently attaches to Ags following activation, where the C3d cleavage fragment can function as a molecular adjuvant to augment humoral immune responses. C3d is proposed to exert its adjuvant-like activities by targeting Ags to the C3d receptor (CD21/35) expressed by B cells and follicular dendritic cells. To directly assess the importance of CD21/35 in mediating the immunostimulatory effects of C3d, CD21/35-deficient (CD21/35−/−) mice were immunized with streptavidin (SA), SA-C3dg tetramers, recombinant HIV gp120 (gp120), or gp120 fused with linear multimers of C3d. Remarkably, SA- and gp120-specific Ab responses were significantly augmented in CD21/35−/− mice when these Ags were complexed with C3d in comparison to Ag alone. In fact, primary and secondary Ab responses and Ab-forming cell responses of CD21/35−/− mice approached those of wild-type mice immunized with SA-C3dg and gp120-C3d. Thus, C3d can function as a molecular adjuvant in the absence of CD21/35 expression.

Potential role for alternatively activated macrophages in the secondary bacterial infection during recovery from influenza.

PURPOSE Secondary bacterial infections are a common complication of influenza. Innate immune host defenses appear to be impaired following influenza, leading to susceptibility to subsequent bacterial infections. Alternatively activated macrophages (AAM) in the lungs may play a critical role in eliciting the hypersusceptibility to secondary bacterial pneumonia. METHODS C57BL6 mice were challenged with sublethal doses of the mouse-adapted A/PR/8/34 (PR8) influenza virus or saline and allowed to recover. At complete recovery (day 14), mice were re-challenged with sublethal doses of Streptococcus pneumoniae serotype 3 (Sp3). RESULTS PR8-recovered mice developed a rapidly fatal pulmonary infection to a 100-fold sublethal pneumococcal challenge, whereas PR8-naive mice demonstrated no mortality or illness. The cytokines which induce AAM (IL-4 and IL-13) and the expression of genes associated with AAM (Arginase-1, FIZZ1, and YM1) were elevated after PR8 infection. Flow cytometry suggests that alveolar macrophages demonstrate the AAM-phenotype, as indicated by MGL-1 and MHCII expression, in response to PR8 infection. Recovery from PR8 was associated with blunted cytokine responses to TLR ligands. CONCLUSIONS The mechanisms of immune regulation during recovery from influenza are being elucidated. We provide evidence that pulmonary AAM are induced during influenza infection and may contribute to the elicitation of hypersusceptibility to a secondary bacterial infection.

Effects of Aging on Influenza Virus Infection Dynamics

ABSTRACT The consequences of influenza virus infection are generally more severe in individuals over 65 years of age (the elderly). Immunosenescence enhances the susceptibility to viral infections and renders vaccination less effective. Understanding age-related changes in the immune system is crucial in order to design prophylactic and immunomodulatory strategies to reduce morbidity and mortality in the elderly. Here, we propose different mathematical models to provide a quantitative understanding of the immune strategies in the course of influenza virus infection using experimental data from young and aged mice. Simulation results suggested a central role of CD8+ T cells for adequate viral clearance kinetics in young and aged mice. Adding the removal of infected cells by natural killer cells did not improve the model fit in either young or aged animals. We separately examined the infection-resistant state of cells promoted by the cytokines alpha/beta interferon (IFN-α/β), IFN-γ, and tumor necrosis factor alpha (TNF-α). The combination of activated CD8+ T cells with any of the cytokines provided the best fits in young and aged animals. During the first 3 days after infection, the basic reproductive number for aged mice was 1.5-fold lower than that for young mice (P < 0.05). IMPORTANCE The fits of our models to the experimental data suggest that the increased levels of IFN-α/β, IFN-γ, and TNF-α (the “inflammaging” state) promote slower viral growth in aged mice, which consequently limits the stimulation of immune cells and contributes to the reported impaired responses in the elderly. A quantitative understanding of influenza virus pathogenesis and its shift in the elderly is the key contribution of this work.

Modeling Influenza Virus Infection: A Roadmap for Influenza Research

Influenza A virus (IAV) infection represents a global threat causing seasonal outbreaks and pandemics. Additionally, secondary bacterial infections, caused mainly by Streptococcus pneumoniae, are one of the main complications and responsible for the enhanced morbidity and mortality associated with IAV infections. In spite of the significant advances in our knowledge of IAV infections, holistic comprehension of the interplay between IAV and the host immune response (IR) remains largely fragmented. During the last decade, mathematical modeling has been instrumental to explain and quantify IAV dynamics. In this paper, we review not only the state of the art of mathematical models of IAV infection but also the methodologies exploited for parameter estimation. We focus on the adaptive IR control of IAV infection and the possible mechanisms that could promote a secondary bacterial coinfection. To exemplify IAV dynamics and identifiability issues, a mathematical model to explain the interactions between adaptive IR and IAV infection is considered. Furthermore, in this paper we propose a roadmap for future influenza research. The development of a mathematical modeling framework with a secondary bacterial coinfection, immunosenescence, host genetic factors and responsiveness to vaccination will be pivotal to advance IAV infection understanding and treatment optimization.

An agent-based model of inflammation and fibrosis following particulate exposure in the lung

Inflammation and airway remodeling occur in a variety of airway diseases. Modeling aspects of the inflammatory and fibrotic processes following repeated exposure to particulate matter may provide insights into a spectrum of airway diseases, as well as prevention/treatment strategies. An agent-based model (ABM) was created to examine the response of an abstracted population of inflammatory cells (nominally macrophages, but possibly including other inflammatory cells such as lymphocytes) and cells involved in remodeling (nominally fibroblasts) to particulate exposure. The model focused on a limited number of relevant interactions, specifically those among macrophages, fibroblasts, a pro-inflammatory cytokine (TNF-α), an anti-inflammatory cytokine (TGF-β1), collagen deposition, and tissue damage. The model yielded three distinct states that were equated with (1) self-resolving inflammation and a return to baseline, (2) a pro-inflammatory process of localized tissue damage and fibrosis, and (3) elevated pro- and anti-inflammatory cytokines, persistent tissue damage, and fibrosis outcomes. Experimental results consistent with these predicted states were observed in histology sections of lung tissue from mice exposed to particulate matter. Systematic in silico studies suggested that the development of each state depended primarily upon the degree and duration of exposure. Thus, a relatively simple ABM resulted in several, biologically feasible, emergent states, suggesting that the model captures certain salient features of inflammation following exposure of the lung to particulate matter. This ABM may hold future utility in the setting of airway disease resulting from inflammation and fibrosis following particulate exposure.

Differential response of the cynomolgus macaque gut microbiota to Shigella infection.

Little is known about the role of gut microbiota in response to live oral vaccines against enteric pathogens. We examined the effect of immunization with an oral live-attenuated Shigella dysenteriae 1 vaccine and challenge with wild-type S. dysenteriae 1 on the fecal microbiota of cynomolgus macaques using 16 S rRNA analysis of fecal samples. Multi-dimensional cluster analysis identified different bacterial community types within macaques from geographically distinct locations. The fecal microbiota of Mauritian macaques, observed to be genetically distinct, harbored a high-diversity community and responded differently to Shigella immunization, as well as challenge compared to the microbiota in non-Mauritian macaques. While both macaque populations exhibited anti-Shigella antibody responses, clinical shigellosis was observed only among non-Mauritian macaques. These studies highlight the importance of further investigation into the possible protective role of the microbiota against enteric pathogens and consideration of host genetic backgrounds in conducting vaccine studies.

Reduction of Anti-HIV-1 Gag Immune Responses During Co-Immunization: Immune Interference by the HIV-1 Envelope

Immunization with more than one immunogen (co-immunization) is an efficient regimen to induce immunity to multiple antigens. However, immune interference has been reported using multi-plasmid DNA immunizations. HIV-1 envelope (Env) and Gag gene products are the predominant immunogens used in current AIDS vaccines, although, few studies have evaluated possible immune interference when these two antigens are co-administered. Therefore, in this study, immune interference during co-inoculation was examined using DNA vaccines expressing lentiviral Envs and Gag from gene sequences optimized for efficient expression in mammalian cells (codon-optimized). BALB/c mice vaccinated in separate hind legs with each plasmid individually elicited high titer immune responses, however, when HIV-1 Env(gp120) and HIV-1 Gag(p55) DNA plasmids were co-inoculated, there was a reduction in the immune responses elicited to HIV-1 Gag(p55). To determine if the anti-HIV-1 Gag(p55) immune interference was specific to HIV-1 Env(gp120), mice were co-immunized with plasmids expressing the surface envelope protein from two additional lentiviruses, Env(gp130)-SIV or Env(gp90)-EIAV, or a soluble form of hemagglutinin (sHA) from influenza virus and HIV-1 Gag(p55)- or SIV Gag(p55)-DNA. Interestingly, there was no reduction in anti-HIV-1 Gag(p55) immune responses using other lentiviral envelopes or the influenza sHA. Also, none of the lentiviral envelopes reduced anti-SIV Gag(p55) immune responses during co-immunization. Therefore, anti-HIV-1 Gag immune interference appears specific to co-immunizations with HIV-1 Env(gp120) and may involve a yet undefined immunological mechanism(s).