Pathogenesis, Humoral Immune Responses, and Transmission between Cohoused Animals in a Ferret Model of Human Respiratory Syncytial Virus Infection.

Small-animal models have been used to obtain many insights regarding the pathogenesis and immune responses induced following infection with human respiratory syncytial virus (hRSV). Among those described to date, infections in cotton rats, mice, guinea pigs, chinchillas, and Syrian hamsters with hRSV strains Long and/or A2 have been well characterized, although clinical isolates have also been examined. Ferrets are also susceptible to hRSV infection, but the pathogenesis and immune responses elicited following infection have not been well characterized. Here, we describe the infection of adult ferrets with hRSV Long or A2 via the intranasal route and characterized virus replication, as well as cytokine induction, in the upper and lower airways. Virus replication and cytokine induction during the acute phase of infection (days 0 to 15 postinfection) were similar between the two strains, and both elicited high levels of F glycoprotein-specific binding and neutralizing antibodies following virus clearance (days 16 to 22 postinfection). Importantly, we demonstrate transmission from experimentally infected donor ferrets to cohoused naive recipients and have characterized virus replication and cytokine induction in the upper airways of infected contact animals. Together, these studies provide a direct comparison of the pathogenesis of hRSV Long and A2 in ferrets and highlight the potential of this animal model to study serological responses and examine interventions that limit transmission of hRSV.IMPORTANCE Ferrets have been widely used to study pathogenesis, immunity, and transmission following human influenza virus infections; however, far less is known regarding the utility of the ferret model to study hRSV infections. Following intranasal infection of adult ferrets with the well-characterized Long or A2 strain of hRSV, we report virus replication and cytokine induction in the upper and lower airways, as well as the development of virus-specific humoral responses. Importantly, we demonstrate transmission of hRSV from experimentally infected donor ferrets to cohoused naive recipients. Together, these findings significantly enhance our understanding of the utility of the ferret as a small-animal model to investigate aspects of hRSV pathogenesis and immunity.

Investigating Viral Interference Between Influenza A Virus and Human Respiratory Syncytial Virus in a Ferret Model of Infection.

Epidemiological studies have observed that the seasonal peak incidence of influenza virus infection is sometimes separate from the peak incidence of human respiratory syncytial virus (hRSV) infection, with the peak incidence of hRSV infection delayed. This is proposed to be due to viral interference, whereby infection with one virus prevents or delays infection with a different virus. We investigated viral interference between hRSV and 2009 pandemic influenza A(H1N1) virus (A[H1N1]pdm09) in the ferret model. Infection with A(H1N1)pdm09 prevented subsequent infection with hRSV. Infection with hRSV reduced morbidity attributed to infection with A(H1N1)pdm09 but not infection, even when an increased inoculum dose of hRSV was used. Notably, infection with A(H1N1)pdm09 induced higher levels of proinflammatory cytokines, chemokines, and immune mediators in the ferret than hRSV. Minimal cross-reactive serological responses or interferon γ-expressing cells were induced by either virus ≥14 days after infection. These data indicate that antigen-independent mechanisms may drive viral interference between unrelated respiratory viruses that can limit subsequent infection or disease.

Evidence for Viral Interference and Cross-reactive Protective Immunity Between Influenza B Virus Lineages.

Background: Two influenza B virus lineages, B/Victoria and B/Yamagata, cocirculate in the human population. While the lineages are serologically distinct, cross-reactive responses to both lineages have been detected. Viral interference describes the situation whereby infection with one virus limits infection and replication of a second virus. We investigated the potential for viral interference between the influenza B virus lineages. Methods: Ferrets were infected and then challenged 3, 10, or 28 days later with pairs of influenza B/Victoria and B/Yamagata viruses. Results: Viral interference occurred at challenge intervals of 3 and 10 days and occasionally at 28 days. At the longer interval, shedding of challenge virus was reduced, and this correlated with cross-reactive interferon γ responses from lymph nodes from virus-infected animals. Viruses from both lineages could prevent or significantly limit subsequent infection with a virus from the other lineage. Coinfections were rare, indicating the potential for reassortment between lineages is limited. Conclusions: These data suggest that innate and cross-reactive immunity mediate viral interference and that this may contribute to the dominance of a specific influenza B virus lineage in any given influenza season. Furthermore, infection with one influenza B virus lineage may be beneficial in protecting against subsequent infection with either influenza B virus lineage.

PRMT1 acts as a suppressor of MHC-I and anti-tumor immunity.

Cancer immunotherapies have demonstrated remarkable success; however, the majority of patients do not respond or develop resistance. Here, we conduct epigenetic gene-targeted CRISPR-Cas9 screens to identify epigenomic factors that limit CD8+ T cell-mediated anti-tumor immunity. We identify that PRMT1 suppresses interferon gamma (Ifnγ)-induced MHC-I expression, thus dampening CD8+ T cell-mediated killing. Indeed, PRMT1 knockout or pharmacological targeting of type I PRMT with the clinical inhibitor GSK3368715 enhances Ifnγ-induced MHC-I expression through elevated STAT1 expression and activation, while re-introduction of PRMT1 in PRMT1-deficient cells reverses this effect. Importantly, loss of PRMT1 enhances the efficacy of anti-PD-1 immunotherapy, and The Cancer Genome Atlas analysis reveals that PRMT1 expression in human melanoma is inversely correlated with expression of human leukocyte antigen molecules, infiltration of CD8+ T cells, and overall survival. Taken together, we identify PRMT1 as a negative regulator of anti-tumor immunity, unveiling clinical type I PRMT inhibitors as immunotherapeutic agents or as adjuncts to existing immunotherapies.

Nucleoprotein of influenza A virus is a major target of immunodominant CD8+ T-cell responses.

Influenza A virus causes annual epidemics and sporadic pandemics, resulting in significant morbidity and mortality worldwide. Vaccines are currently available; however, they induce a non-strain-cross protective humoral immune response directed against the rapidly mutating surface glycoproteins, and thus need to be updated annually. As T cells are directed against more conserved internal influenza proteins, a T-cell-based vaccine has the potential to induce long-lasting and cross-strain protective CD8(+) T-cell immunity, and in that way minimize the severity of influenza infection. However, to rationally design such vaccines, we need to identify immunogenic T-cell regions within the most antigenic viral proteins. In this study, we have used a systematic approach to identify immunodominant peptides in HLA-A2-negative donors. A broad range of CD8(+) T-cell responses were observed and 6/7 donors had an immunodominant response against the relatively conserved internal nucleoprotein (NP). Dissecting the minimal epitope regions within the immunogenic NP led to the identification of six novel immunodominant epitopes, which include a 12-mer and an 8-mer peptides. The majority of immunodominant epitopes was clustered within the carboxyl terminal 2/3 of the NP protein and were highly conserved. We also subjected NP to three common computer algorithms for epitope prediction and found that most of the novel epitopes would not have been predicted. Our study emphasizes the importance of using a systematic approach to identify immunodominant CD8(+) T-cell responses and suggests that the epitope-rich regions within NP present a promising target for the T-cell-mediated multi-strain influenza vaccine.

γδ T Cells in the Tumor Microenvironment-Interactions With Other Immune Cells.

A growing number of studies have shown that γδ T cells play a pivotal role in mediating the clearance of tumors and pathogen-infected cells with their potent cytotoxic, cytolytic, and unique immune-modulating functions. Unlike the more abundant αß T cells, γδ T cells can recognize a broad range of tumors and infected cells without the requirement of antigen presentation via major histocompatibility complex (MHC) molecules. Our group has recently demonstrated parts of the mechanisms of T-cell receptor (TCR)-dependent activation of Vγ9Vδ2+ T cells by tumors following the presentation of phosphoantigens, intermediates of the mevalonate pathway. This process is mediated through the B7 immunoglobulin family-like butyrophilin 2A1 (BTN2A1) and BTN3A1 complexes. Such recognition results in activation, a robust immunosurveillance process, and elicits rapid γδ T-cell immune responses. These include targeted cell killing, and the ability to produce copious quantities of cytokines and chemokines to exert immune-modulating properties and to interact with other immune cells. This immune cell network includes αß T cells, B cells, dendritic cells, macrophages, monocytes, natural killer cells, and neutrophils, hence heavily influencing the outcome of immune responses. This key role in orchestrating immune cells and their natural tropism for tumor microenvironment makes γδ T cells an attractive target for cancer immunotherapy. Here, we review the current understanding of these important interactions and highlight the implications of the crosstalk between γδ T cells and other immune cells in the context of anti-tumor immunity.

Divergent T-cell receptor recognition modes of a HLA-I restricted extended tumour-associated peptide.

Human leukocyte antigen (HLA)-I molecules generally bind short peptides (8-10 amino acids), although extended HLA-I restricted peptides (>10 amino acids) can be presented to T cells. However, the function of such extended HLA-I epitopes in tumour immunity, and how they would be recognised by T-cell receptors (TCR) remains unclear. Here we show that the structures of two distinct TCRs (TRAV4+TRAJ21+-TRBV28+TRBJ2-3+ and TRAV4 + TRAJ8+-TRBV9+TRBJ2-1+), originating from a polyclonal T-cell repertoire, bind to HLA-B*07:02, presenting a 13-amino-acid-long tumour-associated peptide, NY-ESO-160-72. Comparison of the structures reveals that the two TCRs differentially binds NY-ESO-160-72-HLA-B*07:02 complex, and induces differing extent of conformational change of the NY-ESO-160-72 epitope. Accordingly, polyclonal TCR usage towards an extended HLA-I restricted tumour epitope translates to differing TCR recognition modes, whereby extensive flexibility at the TCR-pHLA-I interface engenders recognition.

Safety, immunogenicity and protection of A(H3N2) live attenuated influenza vaccines containing wild-type nucleoprotein in a ferret model.

Live attenuated influenza vaccines (LAIVs) are promising tools for the induction of broad protection from influenza due to their ability to stimulate cross-reactive T cells against influenza pathogens. One of the major targets for cytotoxic T-cell immunity is viral nucleoprotein (NP), which is relatively conserved among antigenically distant influenza viruses. Nevertheless, a diversity of epitope composition has been found in the NP protein of different lineages of influenza A viruses. The H2N2 master donor virus which is currently used as a backbone for the LAIV and donor of the six genomic segments encoding the internal proteins, A/Leningrad/134/17/57 (MDV Len/17), was isolated 60 years ago. As such, NP-specific T-cell immunity induced upon vaccination with classical LAIVs with a 6:2 genome composition containing this older NP might be suboptimal against currently circulating influenza viruses. In this study, a panel of H3N2 LAIV candidates with wild-type NP genes derived from circulating viruses were generated by reverse genetics (5:3 genome composition). These viruses displayed the cold adaptation and temperature sensitivity phenotypes of MDV Len/17 in vitro. LAIVs with both 6:2 and 5:3 genome compositions were attenuated and replicated to a similar extent in the upper respiratory tract of ferrets. LAIVs were immunogenic as high neutralizing and hemagglutination inhibition serum antibody titers were detected 21 days after infection. All vaccinated animals were protected against infection with heterologous H3N2 influenza A viruses. Thus, LAIV with a 5:3 genome composition is safe, immunogenic and can induce cross-protective immunity.

Fine-mapping naturally occurring NY-ESO-1 antibody epitopes in melanoma patients' sera using short overlapping peptides and full-length recombinant protein.

The tumor antigen NY-ESO-1 is one of the most antigenic cancer-testis antigens, first identified by serologic analysis of a recombinant cDNA expression library (SEREX). NY-ESO-1 is expressed in different types of cancers including melanoma. NY-ESO-1-specific spontaneous humoral and cellular immune responses are detected in a large proportion of patients with advanced NY-ESO-1-expressing cancers. Therefore NY-ESO-1 is a good candidate antigen for immunotherapy. Although cellular immune responses to NY-ESO-1 are well characterized, much less is known about the humoral immune responses. In this study, we finely mapped linear antibody epitopes using sera from melanoma patients and shorter overlapping peptide sets. We have shown that melanoma patients' humoral immune systems responded to NY-ESO-1 differently in each individual with widely differing antibody specificity, intensity and antibody subtypes. This knowledge will help us further understand anti-tumor immunity and may also help us to monitor cancer progress and cancer vaccine efficacy in the future.

A novel HLA-B18 restricted CD8+ T cell epitope is efficiently cross-presented by dendritic cells from soluble tumor antigen.

NY-ESO-1 has been a major target of many immunotherapy trials because it is expressed by various cancers and is highly immunogenic. In this study, we have identified a novel HLA-B*1801-restricted CD8(+) T cell epitope, NY-ESO-1(88-96) (LEFYLAMPF) and compared its direct- and cross-presentation to that of the reported NY-ESO-1(157-165) epitope restricted to HLA-A*0201. Although both epitopes were readily cross-presented by DCs exposed to various forms of full-length NY-ESO-1 antigen, remarkably NY-ESO-1(88-96) is much more efficiently cross-presented from the soluble form, than NY-ESO-1(157-165). On the other hand, NY-ESO-1(157-165) is efficiently presented by NY-ESO-1-expressing tumor cells and its presentation was not enhanced by IFN-γ treatment, which induced immunoproteasome as demonstrated by Western blots and functionally a decreased presentation of Melan A(26-35); whereas NY-ESO-1(88-96) was very inefficiently presented by the same tumor cell lines, except for one that expressed high level of immunoproteasome. It was only presented when the tumor cells were first IFN-γ treated, followed by infection with recombinant vaccinia virus encoding NY-ESO-1, which dramatically increased NY-ESO-1 expression. These data indicate that the presentation of NY-ESO-1(88-96) is immunoproteasome dependent. Furthermore, a survey was conducted on multiple samples collected from HLA-B18(+) melanoma patients. Surprisingly, all the detectable responses to NY-ESO-1(88-96) from patients, including those who received NY-ESO-1 ISCOMATRIX™ vaccine were induced spontaneously. Taken together, these results imply that some epitopes can be inefficiently presented by tumor cells although the corresponding CD8(+) T cell responses are efficiently primed in vivo by DCs cross-presenting these epitopes. The potential implications for cancer vaccine strategies are further discussed.