Timothy J. Powell

Preliminary Assessment of the Efficacy of a T-Cell–Based Influenza Vaccine, MVA-NP+M1, in Humans

A single vaccination with MVA-NP+M1 boosts T-cell responses to conserved influenza antigens in humans. Protection against influenza disease and virus shedding was demonstrated in an influenza virus challenge study.

Autophagy is a critical regulator of memory CD8+ T cell formation

During infection, CD8+ T cells initially expand then contract, leaving a small memory pool providing long lasting immunity. While it has been described that CD8+ T cell memory formation becomes defective in old age, the cellular mechanism is largely unknown. Autophagy is a major cellular lysosomal degradation pathway of bulk material, and levels are known to fall with age. In this study, we describe a novel role for autophagy in CD8+ T cell memory formation. Mice lacking the autophagy gene Atg7 in T cells failed to establish CD8+ T cell memory to influenza and MCMV infection. Interestingly, autophagy levels were diminished in CD8+ T cells from aged mice. We could rejuvenate CD8+ T cell responses in elderly mice in an autophagy dependent manner using the compound spermidine. This study reveals a cell intrinsic explanation for poor CD8+ T cell memory in the elderly and potentially offers novel immune modulators to improve aged immunity. DOI: http://dx.doi.org/10.7554/eLife.03706.001

Examination of Influenza Specific T Cell Responses after Influenza Virus Challenge in Individuals Vaccinated with MVA-NP+M1 Vaccine

Current influenza vaccines stimulate neutralising antibody to the haemagglutinin antigen but as there is antigenic drift in HA it is difficult to prepare a vaccine in advance against an emergent strain. A potential strategy is to induce CD8+ and CD4+ T cells that recognize epitopes within internal proteins that are less subject to antigenic drift. Augmenting humoral responses to HA with T cell responses to more conserved antigens may result in a more broadly protective vaccine. In this study, we evaluate the quality of influenza specific T cell responses in a clinical trial using MVA-NP+M1 vaccination followed by influenza virus challenge. In vaccinated volunteers, the expression of Granzyme A, Perforin and CD57 on influenza HLA A*02 M158–66 antigen specific cells was higher than non-vaccinated volunteers before and after challenge despite a similar frequency of antigen specific cells. BCL2 expression was lower in vaccinated volunteers. These data indicate that antigen specific T cells are a useful additional measure for use in human vaccination or immunization studies.

Rat bone marrow-derived dendritic cells, but not ex vivo dendritic cells, secrete nitric oxide and can inhibit T-cell proliferation

The relationships between different dendritic cell (DC) populations are not clearly established. In particular, it is not known how DC generated in vitro relate to those identified in vivo. Here we have characterized rat bone marrow‐derived DC (BMDC) and compared them with DC isolated from spleen (SDC) and pseudo‐afferent lymph (LDC). BMDC express typical DC markers and are mostly OX41 positive and CD4 negative. In contrast to ex vivo DC, some BMDC express Fc receptors. FcR+ and FcR− BMDC express similar levels of major histocompatibility complex class II molecules (MHC) and are B7 positive, but some FcR− BMDC express high levels of B7. In contrast to freshly isolated or cultured ex vivo SDC and LDC, both BMDC subpopulations can express inducible nitric oxide synthase (iNOS) and can secrete nitric oxide (NO) in amounts similar to those secreted by peritoneal macrophages. Despite expressing MHC class II and B7, FcR+ BMDC stimulate only a very weak MLR and inhibit stimulation by FcR− BMDC and ex vivo DC. Inhibition is only partially NO dependent. FcR+ BMDC are not macrophages, as judged by adherence and phagocytosis. Both subpopulations are able to present antigen to primed T cells in vitro and are able to prime naïve CD4 T cells in vivo. However, unlike SDC, BMDC are unable to stimulate cytotoxic T‐lymphocyte (CTL) responses to a minor histocompatibility antigen. Thus, BMDC show marked differences to ex vivo DC and their relationship to those of in vivo DC populations, to date, is unclear.

Focused antibody response to influenza linked to antigenic drift

The selective pressure that drives antigenic changes in influenza viruses is thought to originate from the human immune response. Here, we have characterized the B cell repertoire from a previously vaccinated donor whose serum had reduced neutralizing activity against the recently evolved clade 6B H1N1pdm09 viruses. While the response was markedly polyclonal, 88% of clones failed to recognize clade 6B viruses; however, the ability to neutralize A/USSR/90/1977 influenza, to which the donor would have been exposed in childhood, was retained. In vitro selection of virus variants with representative monoclonal antibodies revealed that a single amino acid replacement at residue K163 in the Sa antigenic site, which is characteristic of the clade 6B viruses, was responsible for resistance to neutralization by multiple monoclonal antibodies and the donor serum. The K163 residue lies in a part of a conserved surface that is common to the hemagglutinins of the 1977 and 2009 H1N1 viruses. Vaccination with the 2009 hemagglutinin induced an antibody response tightly focused on this common surface that is capable of selecting current antigenic drift variants in H1N1pdm09 influenza viruses. Moreover, amino acid replacement at K163 was not highlighted by standard ferret antisera. Human monoclonal antibodies may be a useful adjunct to ferret antisera for detecting antigenic drift in influenza viruses.

Pseudotyped Influenza A Virus as a Vaccine for the Induction of Heterotypic Immunity

ABSTRACT There is a need for vaccines that can protect broadly across all influenza A strains. We have produced a pseudotyped influenza virus based on suppression of the A/PR/8/34 hemagglutinin signal sequence (S-FLU) that can infect cells and express the viral core proteins and neuraminidase but cannot replicate. We show that when given by inhalation to mice, S-FLU is nonpathogenic but generates a vigorous T cell response in the lung associated with markedly reduced viral titers and weight loss after challenge with H1 and H3 influenza viruses. These properties of S-FLU suggest that it may have potential as a broadly protective A virus vaccine, particularly in the setting of a threatened pandemic before matched subunit vaccines become available.

Identification of H5N1-specific T-cell responses in a high-risk cohort in vietnam indicates the existence of potential asymptomatic infections.

BACKGROUND Most reported human H5N1 viral infections have been severe and were detected after hospital admission. A case ascertainment bias may therefore exist, with mild cases or asymptomatic infections going undetected. We sought evidence of mild or asymptomatic H5N1 infection by examining H5N1-specific T-cell and antibody responses in a high-risk cohort in Vietnam. METHODS Peripheral blood mononuclear cells were tested using interferon-γ enzyme-linked immunospot T assays measuring the response to peptides of influenza H5, H3, and H1 hemagglutinin (HA), N1 and N2 neuraminidase, and the internal proteins of H3N2. Horse erythrocyte hemagglutination inhibition assay was performed to detect antibodies against H5N1. RESULTS Twenty-four of 747 individuals demonstrated H5-specific T-cell responses but little or no cross-reactivity with H3 or H1 HA peptides. H5N1 peptide-specific T-cell lines that did not cross-react with H1 or H3 influenza virus HA peptides were generated. Four individuals also had antibodies against H5N1. CONCLUSIONS This is the first report of ex vivo H5 HA-specific T-cell responses in a healthy but H5N1-exposed population. Our results indicate that the presence of H5N1-specific T cells could be an additional diagnostic tool for asymptomatic H5N1 infection.

M1-like monocytes are a major immunological determinant of severity in previously healthy adults with life-threatening influenza

In each influenza season, a distinct group of young, otherwise healthy individuals with no risk factors succumbs to life-threatening infection. To better understand the cause for this, we analyzed a broad range of immune responses in blood from a unique cohort of patients, comprising previously healthy individuals hospitalized with and without respiratory failure during one influenza season, and infected with one specific influenza A strain. This analysis was compared with similarly hospitalized influenza patients with known risk factors (total of n = 60 patients recruited). We found a sustained increase in a specific subset of proinflammatory monocytes, with high TNF-α expression and an M1-like phenotype (independent of viral titers), in these previously healthy patients with severe disease. The relationship between M1-like monocytes and immunopathology was strengthened using murine models of influenza, in which severe infection generated using different models (including the high-pathogenicity H5N1 strain) was also accompanied by high levels of circulating M1-like monocytes. Additionally, a raised M1/M2 macrophage ratio in the lungs was observed. These studies identify a specific subtype of monocytes as a modifiable immunological determinant of disease severity in this subgroup of severely ill, previously healthy patients, offering potential novel therapeutic avenues.In each influenza season, a distinct group of young, otherwise healthy individuals with no risk factors succumbs to life-threatening infection. To better understand the cause for this, we analyzed a broad range of immune responses in blood from a unique cohort of patients, comprising previously healthy individuals hospitalized with and without respiratory failure during one influenza season, and infected with one specific influenza A strain. This analysis was compared with similarly hospitalized influenza patients with known risk factors (total of n = 60 patients recruited). We found a sustained increase in a specific subset of proinflammatory monocytes, with high TNF-α expression and an M1-like phenotype (independent of viral titers), in these previously healthy patients with severe disease. The relationship between M1-like monocytes and immunopathology was strengthened using murine models of influenza, in which severe infection generated using different models (including the high-pathogenicity H5N1 strain) was also accompanied by high levels of circulating M1-like monocytes. Additionally, a raised M1/M2 macrophage ratio in the lungs was observed. These studies identify a specific subtype of monocytes as a modifiable immunological determinant of disease severity in this subgroup of severely ill, previously healthy patients, offering potential novel therapeutic avenues.

Boosted Influenza-Specific T Cell Responses after H5N1 Pandemic Live Attenuated Influenza Virus Vaccination.

Background In a phase I clinical trial, a H5N1 pandemic live attenuated influenza virus (pLAIV) VN2004 vaccine bearing avian influenza H5N1 hemagglutinin (HA) and NA genes on the A/Ann Arbor cold-adapted vaccine backbone displayed very restricted replication. We evaluated T cell responses to H5N1 pLAIV vaccination and assessed pre-existing T cell responses to determine whether they were associated with restricted replication of the H5N1 pLAIV. Method ELISPOT assays were performed using pools of overlapping peptides spanning the entire H5N1 proteome and the HA proteins of relevant seasonal H1N1 and H3N2 viruses. We tested stored peripheral blood mononuclear cells (PBMCs) from 21 study subjects who received two doses of the H5N1 pLAIV. The PBMCs were collected 1 day before and 7 days after the first and second pLAIV vaccine doses, respectively. Result T cell responses to conserved internal proteins M and NP were significantly boosted by vaccination (p = 0.036). In addition, H5N1 pLAIV appeared to preferentially stimulate and boost pre-existing seasonal influenza virus HA-specific T cell responses that showed low cross-reactivity with the H5 HA. We confirmed this observation by T cell cloning and identified a novel HA-specific epitope. However, we did not find any evidence that pre-existing T cells prevented pLAIV replication and take. Conclusion We found that cross-reactive T cell responses could be boosted by pLAIV regardless of the induction of antibody. The impact of the “original antigenic sin” phenomenon in a subset of volunteers, with preferential expansion of seasonal influenza-specific but not H5N1-specific T cell responses merits further investigation.

The Carbomer-Lecithin Adjuvant Adjuplex Has Potent Immunoactivating Properties and Elicits Protective Adaptive Immunity against Influenza Virus Challenge in Mice

ABSTRACT The continued discovery and development of adjuvants for vaccine formulation are important to safely increase potency and/or reduce the antigen doses of existing vaccines and tailor the adaptive immune response to newly developed vaccines. Adjuplex is a novel adjuvant platform based on a purified lecithin and carbomer homopolymer. Here, we analyzed the adjuvant activity of Adjuplex in mice for the soluble hemagglutinin (HA) glycoprotein of influenza A virus. The titration of Adjuplex revealed an optimal dose of 1% for immunogenicity, eliciting high titers of HA-specific IgG but inducing no significant weight loss. At this dose, Adjuplex completely protected mice from an otherwise lethal influenza virus challenge and was at least as effective as the adjuvants monophosphoryl lipid A (MPL) and alum in preventing disease. Adjuplex elicited balanced Th1-/Th2-type immune responses with accompanying cytokines and triggered antigen-specific CD8+ T-cell proliferation. The use of the peritoneal inflammation model revealed that Adjuplex recruited dendritic cells (DCs), monocytes, and neutrophils in the context of innate cytokine and chemokine secretion. Adjuplex neither triggered classical maturation of DCs nor activated a pathogen recognition receptor (PRR)-expressing NF-κB reporter cell line, suggesting a mechanism of action different from that reported for classical pathogen-associated molecular pattern (PAMP)-activated innate immunity. Taken together, these data reveal Adjuplex to be a potent and well-tolerated adjuvant with application for subunit vaccines.