Biology subjects

LaPointe, A. T.

Publications and source records attributed to LaPointe, A. T..

5 recordsLinked to original sources

bioRxiv2026

Discovering Novel Therapeutic VHHs for Emerging Viruses: Perspectives from VEEV Selection Strategies

Evolution or emergence of a new viral variant is a significant public health concern. Alphaviruses, such as Venezuelan equine encephalitis virus (VEEV), are mosquito-borne viruses which are becoming more prevalent due to expansion of vector habitats. The increased prevalence of such viruses provides opportunities for novel variants to evolve. Key therapeutic molecules that could be developed against viral pathogens are recombinant antibodies or antibody fragments, such as the variable heavy domain of heavy chain antibodies (VHHs). These proteins can neutralize or sequester viral particles, preventing or reducing infection. However, due to the evolution of viruses, there is a need to isolate new antibodies and direct their binding to particular epitopes on the virus. In vitro selections offer a promising pathway for the selection of therapeutic antibodies, but as we demonstrate, the choice of a target for these selections is key to obtaining the desired viral binding characteristics. Here we report four novel "human" VHHs which bind to the VEEV E2 protein selected using different strategies that include both computational and biochemical design of suitable antigens and whole virus selections. These VHHs have distinct complementarity-determining regions (CDRs). Multiple VHHs bind to the VEEV viral particles in ELISAs, and we report the peptide epitope recognized by these VHHs. Though non-neutralizing, when immobilized, these VHHs bind to and sequester VEEV viral particles preventing infection, demonstrating the potential of these VHHs to perform viral "sponging". The selection strategies we report may have applications to further antibody developments against other viruses. Significance/ImportanceAlphaviruses, and in particular Venezuelan equine encephalitis virus (VEEV), are recognized for their ability to cause severe disease and for their potential to be used as a biothreat. Despite this, there are currently no antiviral therapies or FDA-approved vaccines available to treat or prevent VEEV infection. This study reports on a novel antibody selection pipeline to produce antibody fragments against VEEV. Antibodies produced via this method showed strong affinity and high specificity to the VEEV E2 glycoprotein in multiple conformations. Additionally, while not neutralizing, the antibody fragments described were shown to be effective as "viral sponges", having the ability to bind, sequester, and remove VEEV virions from solution, which represents a novel therapeutic approach.

immunology↗

bioRxiv2025

Specific and versatile monoclonal antibodies for hantavirus research

Rodent-borne hantaviruses pose a continual public health threat to humans through zoonotic transmission, with case fatality rates of up to 50% in some cases. Human infections can lead to hemorrhagic fever with renal syndrome (HFRS) or hantavirus cardio-pulmonary syndrome (HCPS), depending on the viral species. Despite the morbidity and mortality associated with this family of viruses, no anti-viral therapeutics or vaccines are available to treat and prevent hantavirus disease. The relative shortage of commercially available reagents to study hantavirus infections in vitro and in vivo likely contributes to the challenges in developing viral countermeasures. This report describes the generation of a panel of mouse monoclonal antibodies that collectively recognize the four viral proteins of Seoul virus (Orthohantavirus seoulense), an Old World hantavirus with worldwide distribution and the causative agent of HFRS. We have validated the specificity and versatility of these antibodies against a subset of Old World and New World hantaviruses in assays relying on antigen recognition in denatured or native conformations. We present several antibodies that specifically recognize the Seoul virus nucleoprotein and polymerase protein in Western blotting and immunostaining assays. We also identified three novel antibodies directed against the glycoprotein complex that are capable of binding to the N-terminal glycoprotein of all hantaviruses tested. These antibodies are freely available to all hantavirus researchers to add to the small, but growing, collection of reliable and available reagents to be used to study hantavirus biology, identify novel antiviral compounds, and measure viral prevalence in the laboratory and the field. ImportancePathogenic hantaviruses cause severe hemorrhagic disease and pose a significant public health threat world-wide. Insufficient research into the biology of these viruses has slowed the development of effective direct-acting antivirals and vaccines. Here, we describe the generation and validation of novel, specific monoclonal antibodies for the detection of Seoul virus proteins in vitro. These reagents can be used to fill in critical gaps in knowledge regarding hantavirus entry, protein expression, and particle generation.

microbiology↗

bioRxiv2025

Impact of Species and Cell-type on the RNA Replication Kinetics of Seoul Virus

Hantaviruses are zoonotic, tri-segmented, negative-sense RNA viruses and a significant public health threat. Viral pathogenesis varies between host species, with rodent reservoir infection being asymptomatic and human infection resulting in severe, immune-mediated disease. Viral pathogenesis is highly dependent on virus replication efficiency since it affects the viruss ability to evade detection and determines the magnitude of the host immune response. However, the molecular replication kinetics for hantaviruses remain poorly defined. Therefore, we developed a sense- and segment-specific quantitative real-time PCR (ssqRT-PCR) assay and a SYBR-based qRT-PCR (Sb-qRT-PCR) assay, allowing us to quantify both negative-sense genome levels and total viral RNA synthesis of the small (S), medium (M), and large (L) segments of Seoul virus (SEOV). We then measured total viral RNA and genome accumulation in reservoir rat endothelial cells (RLMVEC), non-reservoir human endothelial cells (HUVEC-C), and Vero E6 epithelial cells. We also measured the ratio of each segment released into the culture supernatant, approximating the relative packaging efficiency. We found that, while the magnitude of viral RNA differed, RNA replication kinetics were largely similar between reservoir and non-reservoir endothelial cells. However, replication and release kinetics differed between infection of endothelial and Vero cells. We also found that the S, M, and L segments were not equally abundant during viral infection or release, but instead followed a trend of M>L>S. Overall, this study validates two qRT-PCR assays to measure SEOV RNA, details the accumulation and release of each viral segment, and demonstrates the impact of host cell type on hantavirus replication. Impact StatementHantavirus infections in humans lead to significant immune-mediated morbidity and mortality around the world and pose a threat to public health. Zoonotic transmission occurs from persistently infected rodents which show no overt signs of disease. Understanding the molecular interactions that drive divergent infection outcomes is incomplete in the field of hantavirus research due to limited tools and reagents. This novel strand- and segment-specific quantitative RT-PCR assay uncovers the unique kinetics of hantavirus genome replication in reservoir and non-reservoir cells. Further, this assay will serve as a valuable tool to investigate antiviral therapies and virus-host interactions critical for polymerase activity.

microbiology↗

bioRxiv2020

Production of Noncapped Genomic RNAs is Critical to Sindbis Virus Disease and Pathogenicity

Alphaviruses are positive-sense RNA viruses that utilize a 5 cap structure to facilitate translation of viral proteins and to protect the viral RNA genome. Nonetheless, significant quantities of viral genomic RNAs that lack a canonical 5 cap structure are produced during alphaviral replication and packaged into viral particles. However, the role/impact of the noncapped genomic RNA (ncgRNA) during alphaviral infection in vivo has yet to be characterized. To determine the importance of the ncgRNA in vivo, the previously described D355A and N376A nsP1 mutations, which increase or decrease nsP1 capping activity respectively, were incorporated into the neurovirulent AR86 strain of Sindbis virus to enable characterization of the impact of altered capping efficiency in a murine model of infection. Mice infected with the N376A nsP1 mutant exhibited slightly decreased rates of mortality and delayed weight loss and neurological symptoms, although levels of inflammation in the brain were similar to wild type infection. The mice infected with the D355A nsP1 mutant showed significantly reduced mortality and morbidity compared to mice infected with wild type virus. Interestingly, both capping mutants had roughly equivalent viral titer in the brain compared to wild type virus, illustrating that the changes in mortality were not due to deficits in viral replication or dissemination. Examination of the brain tissue revealed that mice infected with the D355A capping mutant had significantly reduced cell death and immune cell infiltration compared to the N376A mutant and wild type virus. Finally, expression of proinflammatory cytokines was found to be significantly decreased in mice infected with the D355A mutant, suggesting that capping efficiency and the production of ncgRNA are vital to eliciting pathogenic levels of inflammation. Collectively, these data indicate that the ncgRNA have important roles during alphaviral infection and suggest a novel mechanism by which noncapped viral RNA aid in viral pathogenesis. AUTHOR SUMMARYMosquito transmitted alphaviruses have been the cause of widespread outbreaks of disease which can range from mild illness to lethal encephalitis or severe polyarthritis. In order to successfully replicate, the alphavirus RNA genome needs a 5 cap structure so that the genome can be translated and produce the viral replication machinery. Despite this, a large number of viral genomes produced during infection do not have a 5 cap structure, and their role during infection is unknown. Using mouse models of infection and point mutations in the nsP1 protein of Sindbis virus which alter the amount of noncapped genomic RNA (ncgRNA) produced, we found the decreasing the production of ncgRNA greatly reduced morbidity and mortality as well as proinflammatory cytokine expression, resulting in less tissue-damaging inflammation in the brain. These studies suggest that the ncgRNAs contribute to pathogenesis through the sensing of the ncgRNAs during alphaviral infection and are necessary for the development of severe disease.

microbiology↗

bioRxiv2018

Increasing the Capping Efficiency of the Sindbis Virus nsP1 Protein Negatively Affects Viral Infection

Alphaviruses are arthropod-borne RNA viruses that are capable of causing severe disease and are a significant burden to public health. Alphaviral replication results in the production of both capped and noncapped viral genomic RNAs, which are packaged into virions during the infections of vertebrate and invertebrate cells. However, the roles that the noncapped genomic RNAs (ncgRNAs) play during alphaviral infection have yet to be exhaustively characterized. Here, the importance of the ncgRNAs to alphaviral infection was assessed by using mutants of the nsP1 protein of Sindbis virus (SINV), which altered the synthesis of the ncgRNAs during infection by modulating the proteins capping efficiency. Specifically, point mutants at residues Y286A and N376A decreased capping efficiency, while a point mutant at D355A increased the capping efficiency of the SINV genomic RNA during genuine viral infection. Viral growth kinetics were significantly reduced for the D355A mutant relative to wild type infection, whereas the Y286A and N376A mutants showed modest decreases in growth kinetics. Overall genomic translation and nonstructural protein accumulation was found to correlate with increases and decreases in capping efficiency. However, genomic, minus strand, and subgenomic viral RNA synthesis was largely unaffected by the modulation of alphaviral capping activity. In addition, translation of the subgenomic vRNA was found to be unimpacted by changes in capping efficiency. The mechanism by which decreased presence of ncgRNAs reduced viral growth kinetics was through the impaired production of viral particles. Collectively, these data illustrate the importance of ncgRNAs to viral infection and suggests that they play in integral role in the production of viral progeny.\n\nImportanceAlphaviruses have been the cause of both localized outbreaks and large epidemics of severe disease. Currently, there are no strategies or vaccines which are either safe or effective for preventing alphaviral infection or treating alphaviral disease. This deficit of viable therapeutics highlights the need to better understand the mechanisms behind alphaviral infection in order to develop novel antiviral strategies for alphaviral disease. In particular, this study details a previously uncharacterized aspect of the alphaviral life cycle, the importance of noncapped genomic viral RNAs to alphaviral infection. This offers new insights into the mechanisms of alphaviral replication and the impact of the noncapped genomic RNAs on viral packaging.

microbiology↗