Potent single-domain antibodies that arrest respiratory syncytial virus fusion protein in its prefusion state

Human respiratory syncytial virus (RSV) is the main cause of lower respiratory tract infections in young children. The RSV fusion protein (F) is highly conserved and is the only viral membrane protein that is essential for infection. The prefusion conformation of RSV F is considered the most relevant target for antiviral strategies because it is the fusion-competent form of the protein and the primary target of neutralizing activity present in human serum. Here, we describe two llama-derived single-domain antibodies (VHHs) that have potent RSV-neutralizing activity and bind selectively to prefusion RSV F with picomolar affinity. Crystal structures of these VHHs in complex with prefusion F show that they recognize a conserved cavity formed by two F protomers. In addition, the VHHs prevent RSV replication and lung infiltration of inflammatory monocytes and T cells in RSV-challenged mice. These prefusion F-specific VHHs represent promising antiviral agents against RSV

Human and bovine respiratory syncytial virus vaccine research and development

Human (HRSV) and bovine (BRSV) respiratory syncytial viruses (RSV) are two closely related viruses, which are the most important causative agents of respiratory tract infections of young children and calves, respectively. BRSV vaccines have been available for nearly 2 decades. They probably have reduced the prevalence of RSV infection but their efficacy needs improvement. In contrast, despite decades of research, there is no currently licensed vaccine for the prevention of HRSV disease. Development of a HRSV vaccine for infants has been hindered by the lack of a relevant animal model that develops disease, the need to immunize immunologically immature young infants, the difficulty for live vaccines to find the right balance between attenuation and immunogenicity, and the risk of vaccine-associated disease. During the past 15 years, intensive research into a HRSV vaccine has yielded vaccine candidates, which have been evaluated in animal models and, for some of them, in clinical trials in humans. Recent formulations have focused on subunit vaccines with specific CD4+ Th-1 immune response-activating adjuvants and on genetically engineered live attenuated vaccines. It is likely that different HRSV vaccines and/or combinations of vaccines used sequentially will be needed for the various populations at risk. This review discusses the recent advances in RSV vaccine development

Pre-existing virus-specific CD8+ T-cells provide protection against pneumovirus-induced disease in mice

Pneumoviruses such as pneumonia virus of mice (PVM), bovine respiratory syncytial virus (bRSV) or human (h)RSV are closely related pneumoviruses that cause severe respiratory disease in their respective hosts. It is well-known that T-cell responses are essential in pneumovirus clearance, but pneumovirus-specific T-cell responses also are important mediators of severe immunopathology. In this study we determined whether memory- or pre-existing, transferred virus-specific CD8 + T-cells provide protection against PVM-induced disease. We show that during infection with a sublethal dose of PVM, both natural killer (NK) cells and CD8 + T-cells expand relatively late. Induction of CD8 + T-cell memory against a single CD8 + T-cell epitope, by dendritic cell (DC)-peptide immunization, leads to partial protection against PVM challenge and prevents Th2 differentiation of PVM-induced CD4 T-cells. In addition, adoptively transferred PVM-specific CD8 + T-cells, covering the entire PVM-specific CD8 + T-cell repertoire, provide partial protection from PVM-induced disease. From these data we infer that antigen-specific memory CD8 + T-cells offer significant protection to PVM-induced disease. Thus, CD8 + T-cells, despite being a major cause of PVM-associated pathology during primary infection, may offer promising targets of a protective pneumovirus vaccine

Single-domain antibodies and their formatting to combat viral infections

Since their discovery in the 1990s, single-domain antibodies (VHHs), also known as NanobodiesA (R), have changed the landscape of affinity reagents. The outstanding solubility, stability, and specificity of VHHs, as well as their small size, ease of production and formatting flexibility favor VHHs over conventional antibody formats for many applications. The exceptional ease by which it is possible to fuse VHHs with different molecular modules has been particularly explored in the context of viral infections. In this review, we focus on VHH formats that have been developed to combat viruses including influenza viruses, human immunodeficiency virus-1 (HIV-1), and human respiratory syncytial virus (RSV). Such formats may significantly increase the affinity, half-life, breadth of protection of an antiviral VHH and reduce the risk of viral escape. In addition, VHHs can be equipped with effector functions, for example to guide components of the immune system with high precision to sites of viral infection

Few and Far Between: How HIV May Be Evading Antibody Avidity

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Recent advances and challenges in antigen engineering & vaccine development

textVaccines play a vital role in public health by preventing infectious disease across the globe. Vaccine formulations represent a weakened form of a microbe or toxin that is injected into the human body to elicit an immune response, generating antibodies to protect against a future infection. To this day, it is a challenge to identify and engineer important antigens and epitopes to focus this immune response in a safe and effective manner. The example of Bordetella pertussis is used to highlight the problems and lessons learned in designing a vaccine for this global epidemic. In particular, this review will focus on the advantages and disadvantages of chemical versus genetic detoxification and whole cell versus acellular vaccines in the context of pertussis. The latter part of this review will provide a summary of general strategies, such as epitope mapping and manipulation, synthesis of truncated variants, reverse vaccinology, and structural vaccinology, that have been successful in addressing increasingly complex diseases. Collectively, these techniques provide an invaluable set of tools to focus the immune response by finding and engineering specific antigens and epitopes.Chemical Engineerin

DNA Vaccines Encoding Antigen Targeted to MHC Class II Induce Influenza-Specific CD8+ T Cell Responses, Enabling Faster Resolution of Influenza Disease

Current influenza vaccines are effective but imperfect, failing to cover against emerging strains of virus and requiring seasonal administration to protect against new strains. A key step to improving influenza vaccines is to improve our understanding of vaccine induced protection. Whilst it is clear that antibodies play a protective role, vaccine induced CD8+ T cells can improve protection. To further explore the role of CD8+ T cells we used a DNA vaccine that encodes antigen dimerised to an immune cell targeting module. Immunising CB6F1 mice with the DNA vaccine in a heterologous prime boost regime with the seasonal protein vaccine improved the resolution of influenza disease compared to protein alone. This improved disease resolution was dependent on CD8+ T cells. However, DNA vaccine regimes that induced CD8+ T cells alone were not protective and did not boost the protection provided by protein. The MHC targeting module used was an anti-I-Ed single chain antibody specific to the BALB/c strain of mice. To test the role of MHC targeting we compared the response between BALB/c, C57BL/6 mice and an F1 cross of the two strains (CB6F1). BALB/c mice were protected, C57BL/6 were not and the F1 had an intermediate phenotype; showing that the targeting of antigen is important in the response. Based on these findings, and in agreement with other studies using different vaccines, we conclude that in addition to antibody, inducing a protective CD8 response is important in future influenza vaccines

T Cell Responses during Acute Respiratory Virus Infection

This article is made available for unrestricted research re-use and secondary analysis in any form or by any means with acknowledgement of the original source. These permissions are granted for the duration of the World Health Organization (WHO) declaration of COVID-19 as a global pandemic.The T cell response is an integral and essential part of the host immune response to acute virus infection. Each viral pathogen has unique, frequently nuanced, aspects to its replication, which affects the host response and as a consequence the capacity of the virus to produce disease. There are, however, common features to the T cell response to viruses, which produce acute limited infection. This is true whether virus replication is restricted to a single site, for example, the respiratory tract (RT), CNS etc., or replication is in multiple sites throughout the body. In describing below the acute T cell response to virus infection, we employ acute virus infection of the RT as a convenient model to explore this process of virus infection and the host response. We divide the process into three phases: the induction (initiation) of the response, the expression of antiviral effector activity resulting in virus elimination, and the resolution of inflammation with restoration of tissue homeostasis

Stable isotope tagging of epitopes: a highly selective strategy for the identification of major histocompatibility complex class I-associated peptides induced upon viral infection.

Identification of peptides presented in major histocompatibility complex (MHC) class I molecules after viral infection is of strategic importance for vaccine development. Until recently, mass spectrometric identification of virus-induced peptides was based on comparative analysis of peptide pools isolated from uninfected and virus-infected cells. Here we report on a powerful strategy aiming at the rapid, unambiguous identification of naturally processed MHC class I-associated peptides, which are induced by viral infection. The methodology, stable isotope tagging of epitopes (SITE), is based on metabolic labeling of endogenously synthesized proteins during infection. This is accomplished by culturing virus-infected cells with stable isotope-labeled amino acids that are expected to be anchor residues (i.e. residues of the peptide that have amino acid side chains that bind into pockets lining the peptide-binding groove of the MHC class I molecule) for the human leukocyte antigen allele of interest. Subsequently these cells are mixed with an equal number of non-infected cells, which are cultured in normal medium. Finally peptides are acid-eluted from immunoprecipitated MHC molecules and subjected to two-dimensional nanoscale LC-MS analysis. Virus-induced peptides are identified through computer-assisted detection of characteristic, binomially distributed ratios of labeled and unlabeled molecules. Using this approach we identified novel measles virus and respiratory syncytial virus epitopes as well as infection-induced self-peptides in several cell types, showing that SITE is a unique and versatile method for unequivocal identification of disease-related MHC class I epitopes

Identification of a common HLA-DP4-restricted T-cell epitope in the conserved region of the respiratory syncytial virus G protein

The cellular immune response to respiratory syncytial virus (RSV) is important in both protection and immunopathogenesis. In contrast to HLA class I, HLA class II-restricted RSV-specific T-cell epitopes have not been identified. Here, we describe the generation and characterization of two human RSV-specific CD4(+)-T-cell clones (TCCs) associated with type 0-like cytokine profiles. TCC 1 was specific for the matrix protein and restricted over HLA-DPB1*1601, while TCC 2 was specific for the attachment protein G and restricted over either HLA-DPB1*0401 or -0402. Interestingly, the latter epitope is conserved in both RSV type A and B viruses. Given the high allele frequencies of HLA-DPB1*0401 and -0402 worldwide, this epitope could be widely recognized and boosted by recurrent RSV infections. Indeed, peptide stimulation of peripheral blood mononuclear cells from healthy adults resulted in the detection of specific responses in 8 of 13 donors. Additional G-specific TCCs were generated from three of these cultures, which recognized the identical (n = 2) or almost identical (n = 1) HLA-DP4-restricted epitope as TCC 2. No significant differences were found between the capacities of cell lines obtained from infants with severe (n = 41) or mild (n = 46) RSV lower respiratory tract infections to function as antigen-presenting cells to the G-specific TCCs, suggesting that the severity of RSV disease is not linked to the allelic frequency of HLA-DP4. In conclusion, we have identified an RSV G-specific human T helper cell epitope restricted by the widely expressed HLA class II alleles DPB1*0401 and -0402. Its putative role in protection and/or immunopathogenesis remains to be determined