Therapeutic effect of CT-P59 against SARS-CoV-2 South African variant.

The global circulation of newly emerging variants of SARS-CoV-2 is a new threat to public health due to their increased transmissibility and immune evasion. Moreover, currently available vaccines and therapeutic antibodies were shown to be less effective against new variants, in particular, the South African (SA) variant, termed 501Y.V2 or B.1.351. To assess the efficacy of the CT-P59 monoclonal antibody against the SA variant, we sought to perform as in vitro binding and neutralization assays, and in vivo animal studies. CT-P59 neutralized B.1.1.7 variant to a similar extent as to wild type virus. CT-P59 showed reduced binding affinity against a RBD (receptor binding domain) triple mutant containing mutations defining B.1.351 (K417N/E484K/N501Y) also showed reduced potency against the SA variant in live virus and pseudovirus neutralization assay systems. However, in vivo ferret challenge studies demonstrated that a therapeutic dosage of CT-P59 was able to decrease B.1.351 viral load in the upper and lower respiratory tracts, comparable to that observed for the wild type virus. Overall, although CT-P59 showed reduced in vitro neutralizing activity against the SA variant, sufficient antiviral effect in B.1.351-infected animals was confirmed with a clinical dosage of CT-P59, suggesting that CT-P59 has therapeutic potential for COVID-19 patients infected with SA variant.

The in vitro and in vivo efficacy of CT-P59 against Gamma, Delta and its associated variants of SARS-CoV-2.

The SARS-CoV-2 variant is rapidly spreading across the world and causes to resurge infections. We previously reported that CT-P59 presented its in vivo potency against Beta variants, despite its reduced activity in cell experiments. Yet, it remains uncertain to exert the antiviral effect of CT-P59 on Gamma, Delta and its associated variants (L452R). To tackle this question, we carried out cell tests and animal studies. CT-P59 showed neutralization against Gamma, Delta, Epsilon, and Kappa variants in cells, with reduced susceptibility. The mouse challenge experiments with Gamma and Delta variants substantiated in vivo potency of CT-P59 showing symptom remission and virus abrogation in the respiratory tract. Collectively, cell and animal studies showed that CT-P59 is effective against Gamma and Delta variants infection, hinting that CT-P59 has therapeutic potential for patients infected with Gamma, Delta and its associated variants.

Variation at Extra-epitopic Amino Acid Residues Influences Suppression of Influenza Virus Replication by M158-66 Epitope-Specific CD8+ T Lymphocytes.

Influenza virus-specific CD8+ T lymphocytes (CTLs) contribute to clearance of influenza virus infections and reduce disease severity. Variation at amino acid residues located in or outside CTL epitopes has been shown to affect viral recognition by virus-specific CTLs. In the present study, we investigated the effect of naturally occurring variation at residues outside the conserved immunodominant and HLA*0201-restricted M158-66 epitope, located in the influenza virus M1 protein, on the extent of virus replication in the presence of CTLs specific for the epitope. To this end, we used isogenic viruses with an M1 gene segment derived from either an avian or a human influenza virus, HLA-transgenic human epithelial cells, human T cell clones specific for the M158-66 epitope or a control epitope, and a novel, purposely developed in vitro system to coculture influenza virus-infected cells with T cells. We found that the M gene segment of a human influenza A/H3N2 virus afforded the virus the capacity to replicate better in the presence of M158-66-specific CTLs than the M gene segment of avian viruses. These findings are in concordance with previously observed differential CTL activation, caused by variation at extra-epitopic residues, and may reflect an immune adaptation strategy of human influenza viruses that allows them to cope with potent CTL immunity to the M158-66 epitope in HLA-A*0201-positive individuals, resulting in increased virus replication and shedding and possibly increasing disease severity.IMPORTANCE Influenza viruses are among the leading causes of acute respiratory tract infections. CD8+ T lymphocytes display a high degree of cross-reactivity with influenza A viruses of various subtypes and are considered an important correlate of protection. Unraveling viral immune evasion strategies and identifying signs of immune adaptation are important for defining the role of CD8+ T lymphocytes in affording protection more accurately. Improving our insight into the interaction between influenza viruses and virus-specific CD8+ T lymphocyte immunity may help to advance our understanding of influenza virus epidemiology, aid in risk assessment of potentially pandemic influenza virus strains, and benefit the design of vaccines that induce more broadly protective immunity.

A phase I/IIa immunotherapy trial of HIV-1-infected patients with Tat, Rev and Nef expressing dendritic cells followed by treatment interruption.

In a phase I/IIa clinical trial, 17 HIV-1 infected patients, stable on cART, received 4 vaccinations with autologous dendritic cells electroporated with mRNA encoding Tat, Rev and Nef, after which cART was interrupted. Vaccination was safe and feasible. During the analytical treatment interruption (ATI), no serious adverse events were observed. Ninety-six weeks following ATI, 6/17 patients remained off therapy. Although induced and/or enhanced CD4(+) and CD8(+) T-cell responses specific for the immunogens were observed in most of the patients, we found no correlation with the number of weeks off cART. Moreover, CD4(+) T-cell counts, plasma viral load and the time remaining off cART following ATI did not differ from historical control data. To conclude, the vaccine was safe, well tolerated and resulted in vaccine-specific immune responses. Since no correlation with clinical parameters could be found, these results warrant further research in order to optimize the efficacy of vaccine-induced T-cell responses.

Annual vaccination against influenza virus hampers development of virus-specific CD8⁺ T cell immunity in children.

Infection with seasonal influenza A viruses induces immunity to potentially pandemic influenza A viruses of other subtypes (heterosubtypic immunity). We recently demonstrated that vaccination against seasonal influenza prevented the induction of heterosubtypic immunity against influenza A/H5N1 virus induced by infection with seasonal influenza in animal models, which correlated with the absence of virus-specific CD8(+) T cell responses. Annual vaccination of all healthy children against influenza has been recommended, but the impact of vaccination on the development of the virus-specific CD8(+) T cell immunity in children is currently unknown. Here we compared the virus-specific CD8(+) T cell immunity in children vaccinated annually with that in unvaccinated children. In the present study, we compared influenza A virus-specific cellular and humoral responses of unvaccinated healthy control children with those of children with cystic fibrosis (CF) who were vaccinated annually. Similar virus-specific CD4(+) T cell and antibody responses were observed, while an age-dependent increase of the virus-specific CD8(+) T cell response that was absent in vaccinated CF children was observed in unvaccinated healthy control children. Our results indicate that annual influenza vaccination is effective against seasonal influenza but hampers the development of virus-specific CD8(+) T cell responses. The consequences of these findings are discussed in the light of the development of protective immunity to seasonal and future pandemic influenza viruses.

FATT-CTL assay for detection of antigen-specific cell-mediated cytotoxicity.

Here we describe a flowcytometric assay that measures the defining function of virus-specific cytotoxic T lymphocytes (CTL), i.e., killing viral protein expressing cells. The fluorescent antigen-transfected target cell (FATT)-CTL assay requires no viruses, recombinant viral vectors, or radioactive isotopes to generate CTL target cells that present naturally processed epitopes. It facilitates developing standardized applications in clinical trial settings. Plasmid vectors encoding antigen-green fluorescent protein (GFP) fusion proteins were used directly to nucleofect immortalized B cells or peripheral blood mononuclear cells (PBMCs). Elimination of antigen-GFP expressing cells by cloned CTL, in vitro sensitized PBMC, or ex vivo PBMC was quantified following a 4-18-h coculture period by flowcytometry. This technology successfully detected cell-mediated cytotoxicity in studies involving human PBMC and various viral antigens, including structural proteins of influenza A virus, and structural and nonstructural HIV proteins. Standardized protocols are currently being developed in the framework of a clinical immunotherapy trial in HIV-infected individuals. The FATT-CTL assay principles facilitate standardized flowcytometric detection of antigenic protein-specific cell-mediated cytotoxicity in many different basic research and clinical trial settings. By measuring their defining function, the FATT-CTL assay contributes to a more complete assessment of antigen-specific CTL responses to infection and vaccination.

ViroSpot microneutralization assay for antigenic characterization of human influenza viruses.

The hemagglutination inhibition (HI) assay has been used for the antigenic characterization of influenza viruses for decades. However, the majority of recent seasonal influenza A viruses of the H3N2 subtype has lost the capacity to agglutinate erythrocytes of various species. The hemagglutination (HA) activity of other A(H3N2) strains is generally sensitive to the action of the neuraminidase inhibitor oseltamivir, which indicates that the neuraminidase and not the hemagglutinin is responsible for the HA activity. These findings complicate the antigenic characterization and selection of A(H3N2) vaccine strains, calling for alternative antigenic characterization assays. Here we describe the development and use of the ViroSpot microneutralization (MN) assay as a reliable and robust alternative for the HI assay. Serum neutralization of influenza A(H3N2) reference virus strains and epidemic isolates was determined by automated readout of immunostained cell monolayers, in a format designed to minimize the influence of infectious virus doses on serum neutralization titers. Neutralization of infection was largely independent from rates of viral replication and cell-to-cell transmission, facilitating the comparison of different virus isolates. Other advantages of the ViroSpot MN assay include its relative insensitivity to variation in test dose of infectious virus, automated capture and analyses of residual infection patterns, and compatibility with standardized large scale analyses. Using this assay, a number of epidemic influenza A(H3N2) strains that failed to agglutinate erythrocytes, were readily characterized antigenically.

Ferrets as a Novel Animal Model for Studying Human Respiratory Syncytial Virus Infections in Immunocompetent and Immunocompromised Hosts.

Human respiratory syncytial virus (HRSV) is an important cause of severe respiratory tract disease in immunocompromised patients. Animal models are indispensable for evaluating novel intervention strategies in this complex patient population. To complement existing models in rodents and non-human primates, we have evaluated the potential benefits of an HRSV infection model in ferrets (Mustela putorius furo). Nine- to 12-month-old HRSV-seronegative immunocompetent or immunocompromised ferrets were infected with a low-passage wild-type strain of HRSV subgroup A (105 TCID50) administered by intra-tracheal or intra-nasal inoculation. Immune suppression was achieved by bi-daily oral administration of tacrolimus, mycophenolate mofetil, and prednisolone. Throat and nose swabs were collected daily and animals were euthanized four, seven, or 21 days post-infection (DPI). Virus loads were determined by quantitative virus culture and qPCR. We observed efficient HRSV replication in both the upper and lower respiratory tract. In immunocompromised ferrets, virus loads reached higher levels and showed delayed clearance as compared to those in immunocompetent animals. Histopathological evaluation of animals euthanized 4 DPI demonstrated that the virus replicated in the respiratory epithelial cells of the trachea, bronchi, and bronchioles. These animal models can contribute to an assessment of the efficacy and safety of novel HRSV intervention strategies.

Construction and characterisation of infectious recombinant HIV-1 clones containing CTL epitopes from structural proteins in Nef.

In this study the construction is described of HIV-1 molecular clones in which CTL epitopes from RT or Env late proteins were inserted into the Nef early protein. The ectopic epitopes were efficiently processed from the recombinant Nef proteins, were recognized by their cognate CTL in cytolytic assays, and did not perturb virus replication or viral protein expression in vitro. These recombinant viruses will therefore be an important tool in studying the effect of distinct epitope expression kinetics on the efficiency of CTL-mediated suppression of HIV-1 replication.

HIV-1 evolution in patients undergoing immunotherapy with Tat, Rev, and Nef expressing dendritic cells followed by treatment interruption.

OBJECTIVES: This study aimed to evaluate HIV sequence evolution in whole genes and in CD8 T-cell epitope regions following immunotherapy and subsequent analytical treatment interruption (ATI). A second objective of this study was to analyze associations between vaccine-specific immune responses and epitope mutation rates. DESIGN: HIV-1-infected patients on combined antiretroviral therapy (cART) were subjected to immunotherapy by the administration of an autologous dendritic cell-based therapeutic vaccine expressing Tat, Rev, and Nef and subsequent ATI. METHODS: HIV-1 genes were amplified and sequenced from plasma RNA obtained before initiation of cART as well as during ATI. Control sequences for virus evolution in untreated HIV-1-infected individuals were obtained from the HIV Sequence Database (Los Alamos). CD8 T-cell epitope regions were defined based on literature data and prediction models. HIV-1-specific immune responses were evaluated to analyze their impact on sequence evolution. RESULTS: Viral sequence evolution in the tat, rev, and nef genes of vaccinated patients was similar to that of controls. The number of mutations observed inside and outside CD8 T-cell epitopes was comparable for vaccine-targeted and nontargeted proteins. We found no evidence for an impact of vaccine-induced or enhanced immune responses on the number of mutations inside or outside epitopes. CONCLUSION: Therapeutic vaccination of HIV-1-infected patients with a dendritic cell-based vaccine targeting Tat, Rev, and Nef did not affect virus evolution at the whole gene level nor at the CD8 T-cell epitope level.

Functional T-cell responses generated by dendritic cells expressing the early HIV-1 proteins Tat, Rev and Nef.

The limitations of highly active anti-retroviral therapy (HAART) have necessitated the development of alternative therapeutic strategies. One of the approaches that has gained prominence in recent years is therapeutic vaccination. We decided to assess the capacity of mature dendritic cells, derived from blood monocytes of HIV-1 infected patients, to generate functional T-cell responses. For this purpose, we constructed a chimeric mRNA encoding the proteins Tat, Rev and Nef. The TaReNef encoding information was linked to the HLA class II-targeting sequence of DC-LAMP. Broadly directed HIV-specific CD4(+) and CD8(+) cytotoxic T cells exhibiting a poly-functional cytokine secretion pattern were generated by co-culturing with autologous chimeric mRNA electroporated dendritic cells. Thus, administration of ex vivo generated dendritic cells expressing the early proteins Tat, Rev and Nef might offer a promising approach for therapeutic vaccination in HIV-1 infection.

Fluorescent antigen-transfected target cell cytotoxic T lymphocyte assay for ex vivo detection of antigen-specific cell-mediated cytotoxicity.

Ex vivo detection of virus-specific cytotoxic T lymphocyte (CTL) responses is limited to the use of methods assessing cytokine production, degranulation, or perforin contents of antigen-specific CD8+ T cells. Generally, their cytotoxic activity is detectable only after cultivation. We describe the fluorescent antigentransfected target cellCTL (FATT-CTL) assay, which measures antigen-specific cytotoxicity ex vivo. Target cells were generated by nucleofection with DNA vectors encoding antigengreen fluorescent protein (GFP) fusion proteins. After coculture at various effector : target (E : T) cell ratios, viable and dead GFP-positive cells were quantified by flow cytometry, and antigen-specific target-cell elimination was calculated. The assay was validated with human immunodeficiency virus (HIV) and influenza virusspecific CTL clones and revealed cytotoxicity at lower E : T cell ratios than standard 51Cr-release assays. Moreover, antigen-specific cytotoxicity was detected ex vivo within 1 day in peripheral blood mononuclear cells from HIV-infected individuals. The FATT-CTL assay provides a versatile tool that will advance our understanding of cell-mediated immunity.

The advantage of early recognition of HIV-infected cells by cytotoxic T-lymphocytes.

Accumulating evidence indicates that cytotoxic T-lymphocytes (CTL) play an important role in the clearing of primary and control of chronic human immunodeficiency virus (HIV) infection. Here, we discuss recent findings that indicate that the timing of target cell recognition critically contributes to CTL effectiveness. In this light several problems that have troubled CTL research are discussed. The use of early proteins like Tat and Rev is proposed for future vaccines design.

The choice of antigen for therapeutic immunization against AIDS.

Vaccination with Rev and Tat can induce or boost immune responses that control HIV more effectively than those generated in the context of a natural infection.

Impact of antigen expression kinetics on the effectiveness of HIV-specific cytotoxic T lymphocytes.

Recent studies indicate that the time required for virus-infected cells to become vulnerable for the activity of CTL is of significance for the capacity of CTL to control ongoing viral reproduction. To investigate whether this applies to the effectiveness of HIV-1-specific CTL, we measured virus production in cultures containing CD4(+) T cells inoculated with HIV at low multiplicity of infection, and CTL directed against an early protein, Rev, or a late protein, RT. The Rev-specific CTL prevented at least 2 log(10) more HIV-1 production, in 10 days, than similar numbers of RT-specific CTL. To study how CTL effectiveness depends on variations in the potency of effector functions and kinetics of HIV protein expression, we developed a mathematical model describing CTL-target cell interactions during successive infection cycles. The results show that substantially higher CTL-mediated target cell elimination rates are required to achieve control as there is less time for CTL to act before infected cells release progeny virions. Furthermore, in vitro experiments with HIV recombinant viruses showed that the RT-specific CTL were at least as effective as the Rev-specific CTL, but only if the RT epitope was expressed as part of the early protein Nef. Together these results indicate that CTL control ongoing HIV reproduction more effectively if they are able to recognize infected cells earlier during individual viral replication cycles. This provides rationale for immunization strategies that aim at inducing, boosting or skewing CTL responses to early regulatory proteins in AIDS vaccine development.

Comparison of the efficacy of early versus late viral proteins in vaccination against SIV.

The immune response against early regulatory proteins of simian- and human immunodeficiency virus (SIV, HIV) has been associated with a milder course of infection. Here, we directly compared vaccination with Tat/Rev versus Pol/Gag. Challenge infection with SIVmac32H (pJ5) suggested that vaccination with Tat/Rev induced cellular immune responses that enabled cynomolgus macaques to more efficiently control SIV replication than the vaccine-induced immune responses against Pol/Gag. Vaccination with Tat/Rev resulted in reduced plasma SIV loads compared with control (P=0.058) or Pol/Gag-vaccinated (P=0.089) animals, with undetectable plasma viral loads in two of the four Tat/Rev-vaccinated animals. Therefore, the results warrant further investigation of the early regulatory proteins and their potential for vaccination against HIV.