The Role of Social Contacts and Original Antigenic Sin in Shaping the Age Pattern of Immunity to Seasonal Influenza

Recent serological studies of seasonal influenza A in humans suggest a striking characteristic profile of immunity against age, which holds across different countries and against different subtypes of influenza. For both H1N1 and H3N2, the proportion of the population seropositive to recently circulated strains peaks in school-age children, reaches a minimum between ages 35–65, then rises again in the older ages. This pattern is little understood. Variable mixing between different age classes can have a profound effect on disease dynamics, and is hence the obvious candidate explanation for the profile, but using a mathematical model of multiple influenza strains, we see that age dependent transmission based on mixing data from social contact surveys cannot on its own explain the observed pattern. Instead, the number of seropositive individuals in a population may be a consequence of ‘original antigenic sin’; if the first infection of a lifetime dominates subsequent immune responses, we demonstrate that it is possible to reproduce the observed relationship between age and seroprevalence. We propose a candidate mechanism for this relationship, by which original antigenic sin, along with antigenic drift and vaccination, results in the age profile of immunity seen in empirical studies.     

Estimating the Life Course of Influenza A(H3N2) Antibody Responses from Cross-Sectional Data

The immunity of a host population against specific influenza A strains can influence a number of important biological processes, from the emergence of new virus strains to the effectiveness of vaccination programmes. However, the development of an individual’s long-lived antibody response to influenza A over the course of a lifetime remains poorly understood. Accurately describing this immunological process requires a fundamental understanding of how the mechanisms of boosting and cross-reactivity respond to repeated infections. Establishing the contribution of such mechanisms to antibody titres remains challenging because the aggregate effect of immune responses over a lifetime are rarely observed directly. To uncover the aggregate effect of multiple influenza infections, we developed a mechanistic model capturing both past infections and subsequent antibody responses. We estimated parameters of the model using cross-sectional antibody titres to nine different strains spanning 40 years of circulation of influenza A(H3N2) in southern China. We found that “antigenic seniority” and quickly decaying cross-reactivity were important components of the immune response, suggesting that the order in which individuals were infected with influenza strains shaped observed neutralisation titres to a particular virus. We also obtained estimates of the frequency and age distribution of influenza infection, which indicate that although infections became less frequent as individuals progressed through childhood and young adulthood, they occurred at similar rates for individuals above age 30 y. By establishing what are likely to be important mechanisms driving epochal trends in population immunity, we also identified key directions for future studies. In particular, our results highlight the need for longitudinal samples that are tested against multiple historical strains. This could lead to a better understanding of how, over the course of a lifetime, fast, transient antibody dynamics combine with the longer-term immune responses considered here.     

Association of Anaplasma marginale Strain Superinfection with Infection Prevalence within Tropical Regions

Strain superinfection occurs when a second strain infects a host already infected with and having mounted an immune response to a primary strain. The incidence of superinfection with Anaplasma marginale, a tick-borne rickettsial pathogen of domestic and wild ruminants, has been shown to be higher in tropical versus temperate regions. This has been attributed to the higher prevalence of infection, with consequent immunity against primary strains and thus greater selective pressure for superinfection with antigenically distinct strains. However an alternative explanation would be the differences in the transmitting vector, Dermacentor andersoni in the studied temperate regions and Rhipicephalus microplus in the studied tropical regions. To address this question, we examined two tropical populations sharing the same vector, R. microplus, but with significantly different infection prevalence. Using two separate markers, msp1α (one allele per genome) and msp2 (multiple alleles per genome), there were higher levels of multiple strain infections in the high infection prevalence as compared to the low prevalence population. The association of higher strain diversity with infection prevalence supports the hypothesis that high levels of infection prevalence and consequent population immunity is the predominant driver of strain superinfection.     

Protection from the 2009 H1N1 Pandemic Influenza by an Antibody from Combinatorial Survivor-Based Libraries

Influenza viruses elude immune responses and antiviral chemotherapeutics through genetic drift and reassortment. As a result, the development of new strategies that attack a highly conserved viral function to prevent and/or treat influenza infection is being pursued. Such novel broadly acting antiviral therapies would be less susceptible to virus escape and provide a long lasting solution to the evolving virus challenge. Here we report the in vitro and in vivo activity of a human monoclonal antibody (A06) against two isolates of the 2009 H1N1 pandemic influenza virus. This antibody, which was obtained from a combinatorial library derived from a survivor of highly pathogenic H5N1 infection, neutralizes H5N1, seasonal H1N1 and 2009 “Swine” H1N1 pandemic influenza in vitro with similar potency and is capable of preventing and treating 2009 H1N1 influenza infection in murine models of disease. These results demonstrate broad activity of the A06 antibody and its utility as an anti-influenza treatment option, even against newly evolved influenza strains to which there is limited immunity in the general population.     

Distinct RIG-I and MDA5 signaling by RNA viruses in innate immunity

Alpha/beta interferon immune defenses are essential for resistance to viruses and can be triggered through the actions of the cytoplasmic helicases retinoic acid-inducible gene I (RIG-I) and melanoma differentiation-associated gene 5 (MDA5). Signaling by each is initiated by the recognition of viral products such as RNA and occurs through downstream interaction with the IPS-1 adaptor protein. We directly compared the innate immune signaling requirements of representative viruses of the Flaviviridae, Orthomyxoviridae, Paramyxoviridae, and Reoviridae for RIG-I, MDA5, and interferon promoter-stimulating factor 1 (IPS-1). In cultured fibroblasts, IPS-1 was essential for innate immune signaling of downstream interferon regulatory factor 3 activation and interferon-stimulated gene expression, but the requirements for RIG-I and MDA5 were variable. Each was individually dispensable for signaling triggered by reovirus and dengue virus, whereas RIG-I was essential for signaling by influenza A virus, influenza B virus, and human respiratory syncytial virus. Functional genomics analyses identified cellular genes triggered during influenza A virus infection whose expression was strictly dependent on RIG-I and which are involved in processes of innate or adaptive immunity, apoptosis, cytokine signaling, and inflammation associated with the host response to contemporary and pandemic strains of influenza virus. These results define IPS-1-dependent signaling as an essential feature of host immunity to RNA virus infection. Our observations further demonstrate differential and redundant roles for RIG-I and MDA5 in pathogen recognition and innate immune signaling that may reflect unique and shared biologic properties of RNA viruses whose differential triggering and control of gene expression may impact pathogenesis and infection.     

Multi-Dimensional Measurement of Antibody-Mediated Heterosubtypic Immunity to Influenza

The human immune response to influenza vaccination depends in part on preexisting cross-reactive (heterosubtypic) immunity from previous infection by, and/or vaccination with, influenza strains that share antigenic determinants with the vaccine strains. However, current methods for assessing heterosubtypic antibody responses against influenza, including the hemagglutination-inhibition (HAI) assay and ELISA, are time and labor intensive, and require moderate amounts of serum and reagents. To address these issues we have developed a fluorescent multiplex assay, mPlex-Flu, that rapidly and simultaneously measures strain specific IgG, IgA, and IgM antibodies against influenza hemagglutinin (HA) from multiple viral strains. We cloned, expressed and purified HA proteins from 12 influenza strains, and coupled them to multiplex beads. Assay validation showed that minimal sample volumes (<5 μl of serum) were needed, and the assay had a linear response over a four Log₁₀ range. The assay detected nanogram levels of anti-influenza specific antibodies, had high accuracy and reproducibility, with an average percentage coefficient of variation (%CV) of 9.06 for intra-assay and 12.94 for inter-assay variability. Pre- and post-intramuscular trivalent influenza vaccination levels of virus specific Ig were consistent with HAI titer and ELISA measurements. A significant advantage of the mPLEX-Flu assay over the HAI assay is the ability to perform antigenic cartography, determining the antigenic distances between influenza HA’s, without mathematical correction for HAI data issues. For validation we performed antigenic cartography on 14 different post-influenza infection ferret sera assayed against 12 different influenza HA’s. Results were in good agreement with a phylogenetic tree generated from hierarchical clustering of the genomic HA sequences. This is the first report of the use of a multiplex method for antigenic cartography using ferret sera. Overall, the mPlex-Flu assay provides a powerful tool to rapidly assess the influenza antibody repertoire in large populations and to study heterosubtypic immunity induced by influenza vaccination.     

In vitro generation of influenza-specific polyfunctional CD4+ T cells suitable for adoptive immunotherapy

Background aimsInfluenza viruses cause potentially fatal respiratory infections in stem cell transplant patients. Specific T cells provide long-lived host adaptive immunity to influenza viruses, and the potential for generating such cells for clinical use was investigatedMethodsThe inactivated influenza vaccine (Fluvax) approved for human use was used as the antigen source. Monocyte-derived dendritic cells pulsed with Fluvax were used to stimulate autologous peripheral blood mononuclear cells (PBMC) on days 0 and 7. Cells were expanded with interleukin (IL)-2 from day 7 onwards. Cell numbers and phenotype were assessed on day 21. The presence of influenza virus-specific cells was assessed by cytokine production and proliferative responses following restimulation with influenza antigensResultsOver 21 days of culture, a mean fold increase of 26.3 in cell number was observed (n = 7). Cultures were predominantly effector and central memory CD4⁺ cells, and expressed a phenotype characteristic of activated antigen-specific cells capable of B-cell helper function. Cytotoxic CD4⁺ and CD8⁺ cells specific for influenza and a high percentage of CD4⁺ cells specific for each of three influenza viruses targeted by Fluvax (H1N1, H3N2 and Brisbane viruses) were generated. In addition, T cells expanded when restimulated with antigens derived from influenza viruses.ConclusionsWe have demonstrated a clinically usable method for producing influenza virus-specific T cells that yield high numbers of highly reactive CD4⁺ cells suitable for adoptive immunotherapy. We propose that reconstructing host immunity through adoptive transfer of influenza virus-specific T cells will reduce the frequency of influenza-related deaths in the period of severe immune suppression that follows stem cell transplantation.     

Impaired cellular immune response to injected bacteria after knockdown of ferritin genes in the hard tick Haemaphysalis longicornis

Iron is an indispensable element for most microorganisms, including many pathogenic bacteria. Iron-withholding is a known component of the innate immunity, particularly of vertebrate hosts. Ticks are vectors of multiple pathogens and reports have shown that they naturally harbor several bacterial species. Thus, tick innate immunity must be crucial in limiting bacterial population to tolerable level that will not cause adverse effects. We have previously characterized two types of the iron-binding protein ferritin (HlFER) in the hard tick Haemaphysalis longicornis, known to be a vector of some protozoan parasites and rickettsiae, and showed their antioxidant function and importance in blood feeding and reproduction. Here we examined the possible role of HlFERs in tick immunity against bacterial infection. After silencing Hlfer genes, adult ticks were injected with live enhanced green fluorescence protein-expressing Escherichia coli, and then monitored for survival rate. Hemolymph that included hemocytes was collected for microscopic examination to observe cellular immune response, and for E. coli culture to determine bacterial viability after injection in the ticks. The expression of some antimicrobial peptides in whole ticks was also analyzed by RT-PCR. Hlfer-silenced ticks had a significantly lower survival rate than control ticks after E. coli injection. Greater number of bacteria inside and outside the hemocytes and higher bacterial colony counts after culture with hemolymph were also observed in Hlfer-silenced ticks. However, no difference on the expression of antimicrobial peptides was observed. These results suggest that ferritin molecules might be important in the cellular immune response of ticks to some bacteria.     

Selection of the host for resistance: genetic control of protective immunity to schistosomes

The genetic control of protective immunity to Schistosoma mansoni infection was studied in an attempt to characterize the effector mechanism of attenuated vaccine induced resistance and to define genetic loci regulating this process. Of all inbred mouse strains surveyed, the P and A (A/J) strains developed the lowest levels of resistance to challenge infection after vaccination with irradiated cercariae. Genetic cross experiments indicated that the protective immunity defects of these two strains are controlled by single yet distinct loci unlinked to either the murine H-2 or immunoglobulin heavy chain loci. Vaccinated P and A strain mice were also found to be defective in their capacity to produce activated macrophages which kill schistosome larvae In vitro. In the case of the P strain, an association of this macrophage larvacidal defect with defective vaccine induced resistance was observed in the progeny of p2 genetic crosses. No other congenital defects in humoral or cellular immune response have been detected which genetically correlate with impaired protective immunity. These results support the hypothesis that T-lymphocyte dependent macrophage activation plays a crucial role in the effector mechanism of anti-schistosome immunity. Since P and A strain mice have previously been shown to be defective in their resistance to other infections, it is possible that the loci determining the impaired protective immunity of these strains to schistosomes may play a general role in the regulation of host susceptibility.     

Antigenic distance and cross-immunity, invasibility and coexistence of pathogen strains in an epidemiological model with discrete antigenic space

In models of pathogen interaction and evolution discrete genotypes in the form of bit strings may be mapped to points in a discrete phenotype space based on similarity in antigenic structure. Cross-immunity between strains, that is the reduction in susceptibility to strain A conferred to a host by infection with strain B, can then be defined for pairs of points in the antigenic space by a specified function. Analysis of an SIR type model shows that, if two strains are at equilibrium, the shape of the cross-immunity function has a strong influence on the invasion and coexistence of a third strain and, consequently, the expected evolutionary pathway. A function that is constant except for discontinuities at the end points is expected to result in the accumulation of diversity until a pair of discordant strains occurs that can, depending on parameter values, exclude all other strains. For a function of the form f(h)=h^q, where h is the antigenic distance between two strains, invasion and coexistence is always possible if q≤1 and little antigenic structure is expected in the pathogen population. However, if q>1 invasion and coexistence may be impossible, depending on parameter values, and the pathogen population is expected to show significant antigenic structuring. In addition to illuminating the role of cross-immunity in pathogen evolution, this analysis indicates that the choice of cross-immunity function, the representation of immunity acquired from multiple previous infections and the number of elements used to characterize the antigenic space must be carefully considered in the development and interpretation of more sophisticated models of pathogen dynamics and evolution.     

Heterogeneity in antibody range and the antigenic drift of influenza A viruses

In this paper we explore the consequences of a heterogeneous immune response in individuals on the evolution of a rapidly mutating virus. We show that several features of the incidence and phylogenetic patterns typical of influenza A may be understood in this framework. In our model, limited diversity and rapid drift of the circulating viral strains result from the interplay of two interacting subpopulations with different types of immune response, narrow or broad, upon infection. The subpopulation with the narrow immune response acts as a reservoir where consecutive mutations escape immunity and can persist. Strains with a number of accumulated mutations escape immunity in the other subpopulation as well, causing larger epidemic peaks in the whole population, and reducing strain diversity. Overall, our model produces a modulation of epidemic peak heights and patterns of antigenic drift consistent with reported observations, suggesting an underlying mechanism for the evolutionary epidemiology of influenza, in particular, and other infectious diseases, more generally.     

Sequencing,Annotation, and Characterization of the Influenza Ferret Infectome

Ferrets have become an indispensable tool in the understanding of influenza virus virulence and pathogenesis. Furthermore, ferrets are the preferred preclinical model for influenza vaccine and therapeutic testing. Here we characterized the influenza infectome during the different stages of the infectious process in ferrets with and without prior specific immunity to influenza. RNA from lung tissue and lymph nodes from infected and naïve animals was subjected to next-generation sequencing, followed by de novo data assembly and annotation of the resulting sequences; this process generated a library comprising 13,202 ferret mRNAs. Gene expression profiles during pandemic H1N1 (pdmH1N1) influenza virus infection were analyzed by digital gene expression and solid support microarrays. As expected during primary infection, innate immune responses were triggered in the lung tissue; meanwhile, in the lymphoid tissue, genes encoding antigen presentation and maturation of effector cells of adaptive immunity increased dramatically. After 5 days postinfection, the innate immune gene expression was replaced by the adaptive immune response, which correlates with viral clearance. Reinfection with homologous pandemic influenza virus resulted in a diminished innate immune response, early adaptive immune gene regulation, and a reduction in clinical severity. The fully annotated ferret infectome will be a critical aid to the understanding of the molecular events that regulate disease severity and host-influenza virus interactions among seasonal, pandemic, and highly pathogenic avian influenzas.     

A20 Deficiency in Lung Epithelial Cells Protects against Influenza A Virus Infection

A20 negatively regulates multiple inflammatory signalling pathways. We here addressed the role of A20 in club cells (also known as Clara cells) of the bronchial epithelium in their response to influenza A virus infection. Club cells provide a niche for influenza virus replication, but little is known about the functions of these cells in antiviral immunity. Using airway epithelial cell-specific A20 knockout (A20^AEC-KO) mice, we show that A20 in club cells critically controls innate immune responses upon TNF or double stranded RNA stimulation. Surprisingly, A20^AEC-KO mice are better protected against influenza A virus challenge than their wild type littermates. This phenotype is not due to decreased viral replication. Instead host innate and adaptive immune responses and lung damage are reduced in A20^AEC-KO mice. These attenuated responses correlate with a dampened cytotoxic T cell (CTL) response at later stages during infection, indicating that A20^AEC-KO mice are better equipped to tolerate Influenza A virus infection. Expression of the chemokine CCL2 (also named MCP-1) is particularly suppressed in the lungs of A20^AEC-KO mice during later stages of infection. When A20^AEC-KO mice were treated with recombinant CCL2 the protective effect was abrogated demonstrating the crucial contribution of this chemokine to the protection of A20^AEC-KO mice to Influenza A virus infection. Taken together, we propose a mechanism of action by which A20 expression in club cells controls inflammation and antiviral CTL responses in response to influenza virus infection.     

Induction of heterosubtypic immunity to influenza virus by intranasal immunization

Recovery from live influenza virus infection is known to induce heterosubtypic immunity. In contrast, immunity induced by inactivated vaccines is predominantly subtype specific. In this study, we investigated the heterosubtypic protective immunity induced by inactivated influenza virus. Intranasal immunization of mice with inactivated influenza virus A/PR8 (H1N1) provided complete protection against the homologous virus and a drift virus within the same subtype, A/WSN (H1N1), but not against the heterosubtypic virus A/Philippines (H3N2). However, coadministration of inactivated virus with cholera toxin as an adjuvant conferred complete heterosubtypic protection, without observed illness, even under conditions of CD4⁺ or CD8⁺ T-cell depletion. Analysis of immune correlates prior to challenge and postchallenge indicated that humoral immune responses with cross-neutralizing activity in lungs and in sera play a major role in conferring protective immunity against heterosubtypic challenge. This study has significant implications for developing broadly cross-reactive vaccines against newly emerging pathogenic influenza viruses.     

The Complex Contributions of Genetics and Nutrition to Immunity in Drosophila melanogaster

Both malnutrition and undernutrition can lead to compromised immune defense in a diversity of animals, and “nutritional immunology” has been suggested as a means of understanding immunity and determining strategies for fighting infection. The genetic basis for the effects of diet on immunity, however, has been largely unknown. In the present study, we have conducted genome-wide association mapping in Drosophila melanogaster to identify the genetic basis for individual variation in resistance, and for variation in immunological sensitivity to diet (genotype-by-environment interaction, or GxE). D. melanogaster were reared for several generations on either high-glucose or low-glucose diets and then infected with Providencia rettgeri, a natural bacterial pathogen of D. melanogaster. Systemic pathogen load was measured at the peak of infection intensity, and several indicators of nutritional status were taken from uninfected flies reared on each diet. We find that dietary glucose level significantly alters the quality of immune defense, with elevated dietary glucose resulting in higher pathogen loads. The quality of immune defense is genetically variable within the sampled population, and we find genetic variation for immunological sensitivity to dietary glucose (genotype-by-diet interaction). Immune defense was genetically correlated with indicators of metabolic status in flies reared on the high-glucose diet, and we identified multiple genes that explain variation in immune defense, including several that have not been previously implicated in immune response but which are confirmed to alter pathogen load after RNAi knockdown. Our findings emphasize the importance of dietary composition to immune defense and reveal genes outside the conventional “immune system” that can be important in determining susceptibility to infection. Functional variation in these genes is segregating in a natural population, providing the substrate for evolutionary response to pathogen pressure in the context of nutritional environment.     

CD8+ T cells Are Preferentially Activated during Primary Low Dose Leishmania major Infection but Are Completely Dispensable during Secondary Anti-Leishmania Immunity

We previously showed that CD8⁺ T cells are required for optimal primary immunity to low dose Leishmania major infection. However, it is not known whether immunity induced by low dose infection is durable and whether CD8⁺ T cells contribute to secondary immunity following recovery from low dose infection. Here, we compared primary and secondary immunity to low and high dose L. major infections and assessed the influence of infectious dose on the quality and magnitude of secondary anti-Leishmania immunity. In addition, we investigated the contribution of CD8⁺ T cells in secondary anti-Leishmania immunity following recovery from low and high dose infections. We found that the early immune response to low and high dose infections were strikingly different: while low dose infection preferentially induced proliferation and effector cytokine production by CD8⁺ T cells, high dose infection predominantly induced proliferation and cytokine production by CD4⁺ T cells. This differential activation of CD4⁺ and CD8⁺ T cells by high and low dose infections respectively, was imprinted during in vitro and in vivo recall responses in healed mice. Both low and high dose-infected mice displayed strong infection-induced immunity and were protected against secondary L. major challenge. While depletion of CD4⁺ cells in mice that healed low and high dose infections abolished resistance to secondary challenge, depletion of CD8⁺ cells had no effect. Collectively, our results show that although CD8⁺ T cells are preferentially activated and may contribute to optimal primary anti-Leishmania immunity following low dose infection, they are completely dispensable during secondary immunity.     

Influenza 2009 pandemic: Cellular immunemediated surveillance modulated by TH17 & Tregs

Influenza A virus is a serious public health threat. Most recently the 2009/H1N1 pandemic virus had an inherent ability to evade the host's immune surveillance through genetic drift, shift, and genomic reassortment. Immune characterization of 2009/H1N1 utilized monoclonal antibodies, neutralizing sera, and proteomics. Increased age may have provided some degree of immunity, but vaccines against seasonal influenza viruses seldom yield cross-reactive immunity, exemplified by 2009/H1N1. Nonetheless, about 33% of individuals, over the age of 60, had cross-reactive neutralizing antibodies against 2009/H1N1, whereas only 6-9% young adults had these antibodies. Children characteristically had no detectable immunity against 2009/H1N1. Taken together, these observations suggest some degree of immune transference with at least certain strains of virus that have afflicted the human population in past decades. Because internal influenza proteins may exhibit less antigenic variation, it is possible that prior exposure to diverse strains of influenza virus provide some immunity to novel strains, including the recent pandemic strain (swine-avian A/H1N1). Current trends in immunological studies - specifically the modulation of cellular immune surveillance provided by TH17 and Tregs - also support the need for additional proteomic research for characterizing novel translational evidence-based treatment interventions based on cytokine function to help defeat the virus. Timely and critical research must characterize the impact of genetics and epigenetics of oral and systemic host immune surveillance responses to influenza A virus. The continued development and application of proteomics and gene expression across viral strains and human tissues increases our ability to combat the spread of influenza epidemics and pandemics.     

Model-Based Simulation and Prediction of an Antiviral Strategy against Influenza A Infection

There is a strong need to develop novel strategies in using antiviral agents to efficiently treat influenza infections. Thus, we constructed a rule-based mathematical model that reflects the complicated interactions of the host immunity and viral life cycle and analyzed the key controlling steps of influenza infections. The main characteristics of the pandemic and seasonal influenza strains were estimated using parameter values derived from cells infected with Influenza A/California/04/2009 and Influenza A/NewCaledonia/20/99, respectively. The quantitative dynamics of the infected host cells revealed a more aggressive progression of the pandemic strain than the seasonal strain. The perturbation of each parameter in the model was then tested for its effects on viral production. In both the seasonal and pandemic strains, the inhibition of the viral release (k_C), the reinforcement of viral attachment (k_V), and an increased transition rate of infected cells into activated cells (k_I) exhibited significant suppression effects on the viral production; however, these inhibitory effects were only observed when the numerical perturbations were performed at the early stages of the infection. In contrast, combinatorial perturbations of both the inhibition of viral release and either the reinforcement of the activation of infected cells or the viral attachment exhibited a significant reduction in the viral production even at a later stage of infection. These results suggest that, in addition to blocking the viral release, a combination therapy that also enhances either the viral attachment or the transition of the infected cells might provide an alternative for effectively controlling progressed influenza infection.     

HLA-A*11:01-restricted CD8+ T cell immunity against influenza A and influenza B viruses in Indigenous and non-Indigenous people

HLA-A*11:01 is one of the most prevalent human leukocyte antigens (HLAs), especially in East Asian and Oceanian populations. It is also highly expressed in Indigenous people who are at high risk of severe influenza disease. As CD8⁺ T cells can provide broadly cross-reactive immunity to distinct influenza strains and subtypes, including influenza A, B and C viruses, understanding CD8⁺ T cell immunity to influenza viruses across prominent HLA types is needed to rationally design a universal influenza vaccine and generate protective immunity especially for high-risk populations. As only a handful of HLA-A*11:01-restricted CD8⁺ T cell epitopes have been described for influenza A viruses (IAVs) and epitopes for influenza B viruses (IBVs) were still unknown, we embarked on an epitope discovery study to define a CD8⁺ T cell landscape for HLA-A*11:01-expressing Indigenous and non-Indigenous Australian people. Using mass-spectrometry, we identified IAV- and IBV-derived peptides presented by HLA-A*11:01 during infection. 79 IAV and 57 IBV peptides were subsequently screened for immunogenicity in vitro with peripheral blood mononuclear cells from HLA-A*11:01-expressing Indigenous and non-Indigenous Australian donors. CD8⁺ T cell immunogenicity screening revealed two immunogenic IAV epitopes (A11/PB2_320-331 and A11/PB2_323-331) and the first HLA-A*11:01-restricted IBV epitopes (A11/M_41-49, A11/NS1_186-195 and A11/NP_511-520). The immunogenic IAV- and IBV-derived peptides were >90% conserved among their respective influenza viruses. Identification of novel immunogenic HLA-A*11:01-restricted CD8⁺ T cell epitopes has implications for understanding how CD8⁺ T cell immunity is generated towards IAVs and IBVs. These findings can inform the development of rationally designed, broadly cross-reactive influenza vaccines to ensure protection from severe influenza disease in HLA-A*11:01-expressing individuals.     

An AGM Model for Changes in Complement during Pregnancy: Neutralization of Influenza Virus by Serum Is Diminished in Late Third Trimester

Pregnant women in the third trimester are at increased risk of severe influenza disease relative to the general population, though mechanisms behind this are not completely understood. The immune response to influenza infection employs both complement (C′) and antibody (Ab). The relative contributions of these components to the anti-viral response are difficult to dissect because most humans have pre-existing influenza-specific Abs. We developed the African green monkey (AGM) as a tractable nonhuman primate model to study changes in systemic innate immunity to influenza during pregnancy. Because the AGMs were influenza-naïve, we were able to examine the role of C′ in influenza virus neutralization using serum from non-pregnant animals before and after influenza infection. We determined that serum from naïve AGMs neutralized influenza via C′, while post-infection neutralization did not require C′, suggesting an Ab-mediated mechanism. The latter mimicked neutralization using human serum. Further, we found that ex vivo neutralization of influenza with both naïve and influenza-immune AGM serum occurred by virus particle aggregation and lysis, with immune serum lysing virus at a much higher rate than naïve serum. We hypothesized that the anti-influenza C′ response would diminish late in AGM pregnancy, corresponding with the time when pregnant women suffer increased influenza severity. We found that influenza neutralization capacity is significantly diminished in serum collected late in the third trimester. Strikingly, we found that circulating levels of C3, C3a, and C4 are diminished late in gestation relative to nonpregnant animals, and while neutralization capacity and serum C3a return to normal shortly after parturition, C3 and C4 levels do not. This AGM model system will enable further studies of the role of physiologic and hormonal changes in downregulating C′-mediated anti-viral immunity during pregnancy, and it will permit the identification of therapeutic targets to improve outcomes of influenza virus infection in pregnant women.