Equine Arteritis Virus in Monocytic Cells Suppresses Differentiation and Function of Dendritic Cells.

Equine viral arteritis is an infectious disease of equids caused by equine arteritis virus (EAV), an RNA virus of the family Arteriviridae. Dendritic cells (DC) are important modulators of the immune response with the ability to present antigen to naïve T cells and can be generated in vitro from monocytes (MoDC). DC are important targets for many viruses and this interaction is crucial for the establishment-or rather not-of an anti-viral immunity. Little is known of the effect EAV has on host immune cells, particularly DC. To study the interaction of eqDC with EAV in vitro, an optimized eqMoDC system was used, which was established in a previous study. MoDC were infected with strains of different genotypes and pathogenicity. Virus replication was determined through titration and qPCR. The effect of the virus on morphology, phenotype and function of cells was assessed using light microscopy, flow cytometry and in vitro assays. This study confirms that EAV replicates in monocytes and MoDC. The replication was most efficient in mature MoDC, but variable between strains. Only the virulent strain caused a significant down-regulation of certain proteins such as CD14 and CD163 on monocytes and of CD83 on mature MoDC. Functional studies conducted after infection showed that EAV inhibited the endocytic and phagocytic capacity of Mo and mature MoDC with minimal effect on immature MoDC. Infected MoDC showed a reduced ability to stimulate T cells. Ultimately, EAV replication resulted in an apoptosis-mediated cell death. Thus, EAV evades the host anti-viral immunity both by inhibition of antigen presentation early after infection and through killing infected DC during replication.

Erratum: Parallel Induction of CH505 B Cell Ontogeny-Guided Neutralizing Antibodies and tHIVconsvX Conserved Mosaic-Specific T Cells against HIV-1.

[This corrects the article DOI: 10.1016/j.omtm.2019.06.003.].

Effect of epitope variant co-delivery on the depth of CD8 T cell responses induced by HIV-1 conserved mosaic vaccines.

To stop the HIV-1 pandemic, vaccines must induce responses capable of controlling vast HIV-1 variants circulating in the population as well as those evolved in each individual following transmission. Numerous strategies have been proposed, of which the most promising include focusing responses on the vulnerable sites of HIV-1 displaying the least entropy among global isolates and using algorithms that maximize vaccine match to circulating HIV-1 variants by vaccine cocktails of optimized complementing sequences. In this study, we investigated CD8 T cell responses induced by a bi-valent mosaic of highly conserved HIVconsvX regions delivered by a combination of simian adenovirus ChAdOx1 and poxvirus MVA. We compared partially and fully mono- and bi-valent prime-boost regimens and their ability to elicit T cells recognizing natural epitope variants using an interferon-γ enzyme-linked immunospot (ELISPOT) assay. We used 11 well-defined CD8 T cell epitopes in two mouse haplotypes and, for each epitope, assessed recognition of the two vaccine forms together with the other most frequent epitope variants in the HIV-1 database. We conclude that for the magnitude and depth of epitope recognition, CD8 T cell responses benefitted in most comparisons from the combined bi-valent mosaic and envisage the main advantage of the bi-valent vaccine during its deployment to diverse populations.

Tetravalent Immunogen Assembled from Conserved Regions of HIV-1 and Delivered as mRNA Demonstrates Potent Preclinical T-Cell Immunogenicity and Breadth.

A vaccine will likely be one of the key tools for ending the HIV-1/AIDS epidemic by preventing HIV-1 spread within uninfected populations and achieving a cure for people living with HIV-1. The currently prevailing view of the vaccine field is to introduce protective antibodies, nevertheless, a vaccine to be effective may need to harness protective T cells. We postulated that focusing a T-cell response on the most vulnerable regions of the HIV-1 proteome while maximizing a perfect match between the vaccine and circulating viruses will control HIV-1 replication. We currently use a combination of replication-deficient simian (chimpanzee) adenovirus and poxvirus modified vaccinia virus Ankara to deliver bivalent conserved-mosaic immunogens to human volunteers. Here, we exploit the mRNA platform by designing tetravalent immunogens designated as HIVconsvM, and demonstrate that mRNA formulated in lipid nanoparticles induces potent, broad and polyfunctional T-cell responses in a pre-clinical model. These results support optimization and further development of this vaccine strategy in experimental medicine trials in humans.

Specificity of CD8+ T-Cell Responses Following Vaccination with Conserved Regions of HIV-1 in Nairobi, Kenya.

Sub-Saharan Africa carries the biggest burden of the human immunodeficiency virus type 1 (HIV-1)/AIDS epidemic and is in an urgent need of an effective vaccine. CD8+ T cells are an important component of the host immune response to HIV-1 and may need to be harnessed if a vaccine is to be effective. CD8+ T cells recognize human leukocyte antigen (HLA)-associated viral epitopes and the HLA alleles vary significantly among different ethnic groups. It follows that definition of HIV-1-derived peptides recognized by CD8+ T cells in the geographically relevant regions will critically guide vaccine development. Here, we study fine details of CD8+ T-cell responses elicited in HIV-1/2-uninfected individuals in Nairobi, Kenya, who received a candidate vaccine delivering conserved regions of HIV-1 proteins called HIVconsv. Using 10-day cell lines established by in vitro peptide restimulation of cryopreserved PBMC and stably HLA-transfected 721.221/C1R cell lines, we confirm experimentally many already defined epitopes, for a number of epitopes we define the restricting HLA molecule(s) and describe four novel HLA-epitope pairs. We also identify specific dominance patterns, a promiscuous T-cell epitope and a rescue of suboptimal T-cell epitope induction in vivo by its functional variant, which all together inform vaccine design.

Parallel Induction of CH505 B Cell Ontogeny-Guided Neutralizing Antibodies and tHIVconsvX Conserved Mosaic-Specific T Cells against HIV-1.

The aim of this work was to start collecting information on rational combination of antibody (Ab) and T cell vaccines into single regimens. Two promising candidate HIV-1 vaccine strategies, sequential isolates of CH505 virus Envs developed for initiation of broadly neutralizing antibody lineages and conserved-mosaic tHIVconsvX immunogens aiming to induce effective cross-clade T cell responses, were combined to assess vaccine interactions. These immunogens were delivered in heterologous vector/modality regimens consisting of non-replicating simian (chimpanzee) adenovirus ChAdOx1 (C), non-replicating poxvirus MVA (M), and adjuvanted protein (P). Outbred CD1-SWISS mice were vaccinated intramuscularly using either parallel CM8M (tHIVconsvX)/CPPP (CH505) or sequential CM16M (tHIVconsvX)/CPPP (CH505) protocols, the latter of which delivered T cell CM prior to the CH505 Env. CM8M (tHIVconsvX) and CPPP or CMMP (CH505) vaccinations alone were included as comparators. The vaccine-elicited HIV-1-specific trimer-binding and neutralizing Abs and CD8+/CD4+ T cell responses induced by the combined and comparator regimens were not statistically separable among regimens. The Ab-lineage immunogen strategy was particularly suited for combined regimens for its likely less potent induction of Env-specific T cell responses relative to homologous epitope-based vaccine strategies. These results inform design of the first rationally combined Ab and T cell vaccine regimens in human volunteers.

Priming With Recombinant BCG Expressing Novel HIV-1 Conserved Mosaic Immunogens and Boosting With Recombinant ChAdOx1 Is Safe, Stable, and Elicits HIV-1-Specific T-Cell Responses in BALB/c Mice.

BCG is currently the only licensed vaccine against tuberculosis (TB) and confers protection against meningitis and miliary tuberculosis in infants, although pulmonary disease protection in adults is inconsistent. Recently, promising HIV-1 immunogens were developed, such as the T-cell immunogens "tHIVconsvX," designed using functionally conserved protein regions across group M strains, with mosaic immunogens to improve HIV-1 variant match and response breadth. In this study, we constructed an integrative E. coli-mycobacterial shuttle plasmid, p2auxo.HIVconsvXint, expressing the immunogens HIVconsv1&2. This expression vector used an antibiotic resistance-free mechanism for plasmid selection and maintenance. It was first transformed into a glycine auxotrophic E. coli strain and subsequently transformed into a lysine auxotrophic Mycobacterium bovis BCG strain to generate vaccines BCG.HIVconsv12auxo.int and BCG.HIVconsv22auxo.int. The DNA sequence coding for the HIVconsv1&2 immunogens and protein expression were confirmed and working vaccine stocks were genetically and phenotypically characterized. We demonstrated that BCG.HIVconsv1&22auxo.int in combination with ChAdOx1.tHIVconsv5&6 were well tolerated and induced HIV-1-specific T-cell responses in adult BALB/c mice. In addition, we showed that the BCG.HIVconsv1&22auxo.int vaccine strains were stable in vitro after 35 bacterial generations and in vivo 7 weeks after inoculation. The use of integrative expression vectors and novel HIV-1 immunogens are likely to have improved the mycobacterial vaccine stability and specific immunogenicity and may enable the development of a useful vaccine platform for priming protective responses against HIV-1/TB and other prevalent pediatric pathogens shortly following birth.

Complete protection of the BALB/c and C57BL/6J mice against Ebola and Marburg virus lethal challenges by pan-filovirus T-cell epigraph vaccine.

There are a number of vaccine candidates under development against a small number of the most common outbreak filoviruses all employing the virus glycoprotein (GP) as the vaccine immunogen. However, antibodies induced by such GP vaccines are typically autologous and limited to the other members of the same species. In contrast, T-cell vaccines offer a possibility to design a single pan-filovirus vaccine protecting against all known and even likely existing, but as yet unencountered members of the family. Here, we used a cross-filovirus immunogen based on conserved regions of the filovirus nucleoprotein, matrix and polymerase to construct simian adenovirus- and poxvirus MVA-vectored vaccines, and in a proof-of-concept study demonstrated a protection of the BALB/c and C57BL/6J mice against high, lethal challenges with Ebola and Marburg viruses, two distant members of the family, by vaccine-elicited T cells in the absence of GP antibodies.

Efficient Induction of T Cells against Conserved HIV-1 Regions by Mosaic Vaccines Delivered as Self-Amplifying mRNA.

Focusing T cell responses on the most vulnerable parts of HIV-1, the functionally conserved regions of HIV-1 proteins, is likely a key prerequisite for vaccine success. For a T cell vaccine to efficiently control HIV-1 replication, the vaccine-elicited individual CD8+ T cells and as a population have to display a number of critical traits. If any one of these traits is suboptimal, the vaccine is likely to fail. Fine-tuning of individual protective characteristics of T cells will require iterative stepwise improvements in clinical trials. Although the second-generation tHIVconsvX immunogens direct CD8+ T cells to predominantly protective and conserved epitopes, in the present work, we have used formulated self-amplifying mRNA (saRNA) to deliver tHIVconsvX to the immune system. We demonstrated in BALB/c and outbred mice that regimens employing saRNA vaccines induced broadly specific, plurifunctional CD8+ and CD4+ T cells, which displayed structured memory subpopulations and were maintained at relatively high frequencies over at least 22 weeks post-administration. This is one of the first thorough analyses of mRNA vaccine-elicited T cell responses. The combination of tHIVconsvX immunogens and the highly versatile and easily manufacturable saRNA platform may provide a long-awaited opportunity to define and optimize induction of truly protective CD8+ T cell parameters in human volunteers.

Randomized phase I trial HIV-CORE 003: Depletion of serum amyloid P component and immunogenicity of DNA vaccination against HIV-1.

BACKGROUND: The failure of DNA vaccination in humans, in contrast to its efficacy in some species, is unexplained. Observational and interventional experimental evidence suggests that DNA immunogenicity may be prevented by binding of human serum amyloid P component (SAP). SAP is the single normal DNA binding protein in human plasma. The drug (R)-1-[6-[(R)-2-carboxypyrrolidin-1-yl]-6-oxo-hexanoyl]pyrrolidine-2-carboxylic acid (CPHPC, miridesap), developed for treatment of systemic amyloidosis and Alzheimer's disease, depletes circulating SAP by 95-99%. The proof-of-concept HIV-CORE 003 clinical trial tested whether SAP depletion by CPHPC would enhance the immune response in human volunteers to DNA vaccination delivering the HIVconsv immunogen derived from conserved sub-protein regions of HIV-1. METHODS: Human volunteers received 3 intramuscular immunizations with an experimental DNA vaccine (DDD) expressing HIV-1-derived immunogen HIVconsv, with or without prior depletion of SAP by CPHPC. All subjects were subsequently boosted by simian (chimpanzee) adenovirus (C)- and poxvirus MVA (M)-vectored vaccines delivering the same immunogen. After administration of each vaccine modality, the peak total magnitudes, kinetics, functionality and memory subsets of the T-cell responses to HIVconsv were thoroughly characterized. RESULTS: No differences were observed between the CPHPC treated and control groups in any of the multiple quantitative and qualitative parameters of the T-cell responses to HIVconsv, except that after SAP depletion, there was a statistically significantly greater breadth of T-cell specificities, that is the number of recognized epitopes, following the DDDC vaccination. CONCLUSIONS: The protocol used here for SAP depletion by CPHPC prior to DNA vaccination produced only a very modest suggestion of enhanced immunogenicity. Further studies will be required to determine whether SAP depletion might have a practical value in DNA vaccination for other plasmid backbones and/or immunogens. TRIAL REGISTRATION: Clinicaltrials.gov NCT02425241.

Long-term follow up of human T-cell responses to conserved HIV-1 regions elicited by DNA/simian adenovirus/MVA vaccine regimens.

BACKGROUND: Durability of vaccine-elicited immune responses is one of the key determinants for vaccine success. Our aim is to develop a vaccination strategy against the human immunodeficiency virus type 1 (HIV-1), which induces protective and durable CD8+ T-cell responses. The central theorem of our approach is to focus T cells on highly conserved regions of the HIV-1 proteome and this is achieved through the use of the first-generation conserved vaccine immunogen HIVconsv. This immunogen vectored by plasmid DNA, simian adenovirus and poxvirus MVA was tested in healthy, HIV-1-negative adults in UK and induced high magnitudes of HIVconsv-specific plurifunctional CD8+ T cells capable of in vitro HIV-1 inhibition. Here, we assessed the durability of these responses. METHODS: Vaccine recipients in trial HIV-CORE 002 were invited to provide a blood sample at 1 and 2 years after vaccination. Their PBMCs were tested in IFN-γ ELISPOT, 25-analyte Luminex, CFSE proliferation and intracellular cytokine staining assays, the last enhanced by HLA-peptide dextramer analysis. RESULTS: 12/12 (1 year) and 8/8 (2 years) returning subjects had median (range) of 990 (150-2495) and 763 (70-1745) IFN-γ SFU/106 PBMC specific for HIVconsv, respectively, and recognized 5 (1-6) out of 6 peptide pools at 2 years. Over one-half of the HIVconsv-specific cells expressed at least 3 functions IFN-γ, TNF-α and CD107a, and were capable of proliferation. Among dextramer-reactive cells, naïve, transitional, effector and terminally differentiated memory subsets were similarly represented. CONCLUSIONS: First generation HIVconsv vaccine induced human T cells, which were plurifunctional and persisted for at least 2 years. TRIAL REGISTRATION: ClinicalTrials.gov NCT01151319.

Influenza A Virus Challenge Models in Cynomolgus Macaques Using the Authentic Inhaled Aerosol and Intra-Nasal Routes of Infection.

Non-human primates are the animals closest to humans for use in influenza A virus challenge studies, in terms of their phylogenetic relatedness, physiology and immune systems. Previous studies have shown that cynomolgus macaques (Macaca fascicularis) are permissive for infection with H1N1pdm influenza virus. These studies have typically used combined challenge routes, with the majority being intra-tracheal delivery, and high doses of virus (> 107 infectious units). This paper describes the outcome of novel challenge routes (inhaled aerosol, intra-nasal instillation) and low to moderate doses (103 to 106 plaque forming units) of H1N1pdm virus in cynomolgus macaques. Evidence of virus replication and sero-conversion were detected in all four challenge groups, although the disease was sub-clinical. Intra-nasal challenge led to an infection confined to the nasal cavity. A low dose (103 plaque forming units) did not lead to detectable infectious virus shedding, but a 1000-fold higher dose led to virus shedding in all intra-nasal challenged animals. In contrast, aerosol and intra-tracheal challenge routes led to infections throughout the respiratory tract, although shedding from the nasal cavity was less reproducible between animals compared to the high-dose intra-nasal challenge group. Intra-tracheal and aerosol challenges induced a transient lymphopaenia, similar to that observed in influenza-infected humans, and greater virus-specific cellular immune responses in the blood were observed in these groups in comparison to the intra-nasal challenge groups. Activation of lung macrophages and innate immune response genes was detected at days 5 to 7 post-challenge. The kinetics of infection, both virological and immunological, were broadly in line with human influenza A virus infections. These more authentic infection models will be valuable in the determination of anti-influenza efficacy of novel entities against less severe (and thus more common) influenza infections.

Gammaherpesvirus infection modulates the temporal and spatial expression of SCGB1A1 (CCSP) and BPIFA1 (SPLUNC1) in the respiratory tract.

Murine γ-herpesvirus 68 (MHV-68) infection of Mus musculus-derived strains of mice is an established model of γ-herpesvirus infection. We have previously developed an alternative system using a natural host, the wood mouse (Apodemus sylvaticus), and shown that the MHV-68 M3 chemokine-binding protein contributes significantly to MHV-68 pathogenesis. Here we demonstrate in A. sylvaticus using high-density micro-arrays that M3 influences the expression of genes involved in the host response including Scgb1a1 and Bpifa1 that encode potential innate defense proteins secreted into the respiratory tract. Further analysis of MHV-68-infected animals showed that the levels of both protein and RNA for SCGB1A1 and BPIFA1 were decreased at day 7 post infection (p.i.) but increased at day 14 p.i. as compared with M3-deficient and mock-infected animals. The modulation of expression was most pronounced in bronchioles but was also present in the bronchi and trachea. Double staining using RNA in situ hybridization and immunohistology demonstrated that much of the BPIFA1 expression occurs in club cells along with SCGB1A1 and that BPIFA1 is stored within granules in these cells. The increase in SCGB1A1 and BPIFA1 expression at day 14 p.i. was associated with the differentiation of club cells into mucus-secreting cells. Our data highlight the role of club cells and the potential of SCGB1A1 and BPIFA1 as innate defense mediators during respiratory virus infection.

Differentiation and activation of equine monocyte-derived dendritic cells are not correlated with CD206 or CD83 expression.

Dendritic cells (DC) are the main immune mediators inducing primary immune responses. DC generated from monocytes (MoDC) are a model system to study the biology of DC in vitro, as they represent inflammatory DC in vivo. Previous studies on the generation of MoDC in horses indicated that there was no distinct difference between immature and mature DC and that the expression profile was distinctly different from humans, where CD206 is expressed on immature MoDC whereas CD83 is expressed on mature MoDC. Here we describe the kinetics of equine MoDC differentiation and activation, analysing both phenotypic and functional characteristics. Blood monocytes were first differentiated with equine granulocyte-macrophage colony-stimulating factor and interleukin-4 generating immature DC (iMoDC). These cells were further activated with a cocktail of cytokines including interferon-γ) but not CD40 ligand to obtain mature DC (mMoDC). To determine the expression of a broad range of markers for which no monoclonal antibodies were available to analyse the protein expression, microarray and quantitative PCR analysis were performed to carry out gene expression analysis. This study demonstrates that equine iMoDC and mMoDC can be distinguished both phenotypically and functionally but the expression pattern of some markers including CD206 and CD83 is dissimilar to the human system.