Treatment of highly virulent mammarenavirus infections-status quo and future directions.

INTRODUCTION: Mammarenaviruses are negative-sense bisegmented enveloped RNA viruses that are endemic in Africa, the Americas, and Europe. Several are highly virulent, causing acute human diseases associated with high case fatality rates, and are considered to be significant with respect to public health impact or bioterrorism threat. AREAS COVERED: This review summarizes the status quo of treatment development, starting with drugs that are in advanced stages of evaluation in early clinical trials, followed by promising candidate medical countermeasures emerging from bench analyses and investigational animal research. EXPERT OPINION: Specific therapeutic treatments for diseases caused by mammarenaviruses remain limited to the off-label use of ribavirin and transfusion of convalescent sera. Progress in identifying novel candidate medical countermeasures against mammarenavirus infection has been slow in part because of the biosafety and biosecurity requirements. However, novel methodologies and tools have enabled increasingly efficient high-throughput molecular screens of regulatory-agency-approved small-molecule drugs and led to the identification of several compounds that could be repurposed for the treatment of infection with several mammarenaviruses. Unfortunately, most of them have not yet been evaluated in vivo. The most promising treatment under development is a monoclonal antibody cocktail that is protective against multiple lineages of the Lassa virus in nonhuman primate disease models.

Influenza virus immune imprinting dictates the clinical outcomes in ferrets challenged with highly pathogenic avian influenza virus H5N1.

Zoonotic transmission of H5N1 highly pathogenic avian influenza virus (HPAIV) into the human population is an increasing global threat. The recent 2022 HPAIV outbreak significantly highlighted this possibility, increasing concern in the general population. The clinical outcomes of H5N1 influenza virus exposure can be determined by an individual's primary influenza virus infection (imprinting) or vaccination status. Immunological imprinting with Group 1 - (H1N1, H2N2, and H2N3) increases survival rates following H5N1 viral infection compared to Group 2 - (H3N2) imprinted individuals. Vaccination against H5N1 influenza viruses can offer protection to at-risk populations; however, stockpiled inactivated H5N1 influenza vaccines are not readily available to the public. We hypothesize that the immunological response to vaccination and subsequent clinical outcome following H5N1 influenza virus infection is correlated with the immunological imprinting status of an individual. To test this hypothesis, our lab established a ferret pre-immune model of disease. Naïve ferrets were intranasally inoculated with seasonal influenza viruses and allowed to recover for 84 days prior to H5N1 virus infection. Ferrets imprinted following H1N1 and H2N3 virus infections were completely protected against lethal H5N1 influenza virus challenge (100% survival), with few to no clinical symptoms. In comparison, H3N2 influenza virus-imprinted ferrets had severe clinical symptoms, delayed disease progression, and a sublethal phenotype (40% mortality). Consecutive infections with H1N1 influenza viruses followed by an H3N2 influenza virus infection did not abrogate the immune protection induced by the original H1N1 influenza virus infection. In addition, ferrets consecutively infected with H1N1 and H2N3 viruses had no clinical symptoms or weight loss. H3N2 pre-immune ferrets were vaccinated with a broadly reactive H5 HA-based or H1 NA-based vaccine (Hu-CO 2). These ferrets were protected against H5N1 influenza virus challenge, whereas ferrets vaccinated with the H1N1 wild-type CA/09 rHA vaccine had similar phenotypes as non-vaccinated H3N2-imprinted ferrets with 40% survival. Overall, Group 2 imprinted ferrets, which were vaccinated with heterologous Group 1 HA vaccines, had redirected immune responses to Group 1 influenza viral antigens and rescued a sublethal phenotype to complete protection.

MVA vector expression of SARS-CoV-2 spike protein and protection of adult Syrian hamsters against SARS-CoV-2 challenge.

Numerous vaccine candidates against SARS-CoV-2, the causative agent of the COVID-19 pandemic, are under development. The majority of vaccine candidates to date are designed to induce immune responses against the viral spike (S) protein, although different forms of S antigen have been incorporated. To evaluate the yield and immunogenicity of different forms of S, we constructed modified vaccinia virus Ankara (MVA) vectors expressing full-length S (MVA-S), the RBD, and soluble S ectodomain and tested their immunogenicity in dose-ranging studies in mice. All three MVA vectors induced spike-specific immunoglobulin G after one subcutaneous immunization and serum titers were boosted following a second immunization. The MVA-S and MVA-ssM elicited the strongest neutralizing antibody responses. In assessing protective efficacy, MVA-S-immunized adult Syrian hamsters were challenged with SARS-CoV-2 (USA/WA1/2020). MVA-S-vaccinated hamsters exhibited less severe manifestations of atypical pneumocyte hyperplasia, hemorrhage, vasculitis, and especially consolidation, compared to control animals. They also displayed significant reductions in gross pathology scores and weight loss, and a moderate reduction in virus shedding was observed post challenge in nasal washes. There was evidence of reduced viral replication by in situ hybridization, although the reduction in viral RNA levels in lungs and nasal turbinates did not reach significance. Taken together, the data indicate that immunization with two doses of an MVA vector expressing SARS-CoV-2 S provides protection against a stringent SARS-CoV-2 challenge of adult Syrian hamsters, reaffirm the utility of this animal model for evaluating candidate SARS-CoV-2 vaccines, and demonstrate the value of an MVA platform in facilitating vaccine development against SARS-CoV-2.

Next-Generation Computationally Designed Influenza Hemagglutinin Vaccines Protect against H5Nx Virus Infections.

H5N1 COBRA hemagglutinin (HA) sequences, termed human COBRA-2 HA, were constructed through layering of HA sequences from viruses isolated from humans collected between 2004-2007 using only clade 2 strains. These COBRA HA proteins, when expressed on the surface of virus-like particles (VLP), elicited protective immune responses in mice, ferrets, and non-human primates. However, these vaccines were not as effective at inducing neutralizing antibodies against newly circulating viruses. Therefore, COBRA HA-based vaccines were updated in order to elicit protective antibodies against the current circulating clades of H5Nx viruses. Next-generation COBRA HA vaccines were designed to encompass the newly emerging viruses circulating in wild avian populations. HA amino acid sequences from avian and human H5 influenza viruses isolated between 2011-2017 were downloaded from the GISAID (Global Initiative on Sharing All Influenza Data). Mice were vaccinated with H5 COBRA rHA that elicited antibodies with hemagglutinin inhibition (HAI) activity against H5Nx viruses from five clades. The H5 COBRA rHA vaccine, termed IAN8, elicited protective immune responses against mice challenged with A/Sichuan/26621/2014 and A/Vietnam/1203/2004. This vaccine elicited antibodies with HAI activity against viruses from clades 2.2, 2.3.2.1, 2.3.4.2, 2.2.1 and 2.2.2. Lungs from vaccinated mice had decreased viral titers and the levels of cellular infiltration in mice vaccinated with IAN-8 rHA were similar to mice vaccinated with wild-type HA comparator vaccines or mock vaccinated controls. Overall, these next-generation H5 COBRA HA vaccines elicited protective antibodies against both historical H5Nx influenza viruses, as well as currently circulating clades of H5N1, H5N6, and H5N8 influenza viruses.

SARS-CoV-2 B.1.1.7 Infection of Syrian Hamster Does Not Cause More Severe Disease, and Naturally Acquired Immunity Confers Protection.

Epidemiological studies have revealed the emergence of multiple severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants of concern (VOC), including the lineage B.1.1.7 that is rapidly replacing old variants. The B.1.1.7 variant has been linked to increased morbidity rates, transmissibility, and potentially mortality. To assess viral fitness in vivo and to address whether the B.1.1.7 variant is capable of immune escape, we conducted infection and reinfection studies in naive and convalescent Syrian hamsters (>10 months old). Nasal wash samples from hamsters infected by a B.1.1.7 variant exhibited slightly higher viral RNA levels but lower infectious titers than those from B.1 (G614) variant-infected hamsters, and the two variants induced comparable lung pathologies in hamsters. Despite a sporadic and transient low-level infection in the nasal cavity, convalescent hamsters that had recovered from a previous USA-WA1 isolate (D614) infection displayed no observable clinical signs or lung pathology following B.1.1.7 rechallenge. Altogether, our study did not find that the B.1.1.7 variant significantly differs from the B.1 variant in pathogenicity in Syrian hamsters and that a heterologous natural infection-induced immunity confers protection against a secondary challenge by the B1.1.7 variant. IMPORTANCE The rapid emergence of several variants of concern of SARS-CoV-2 calls for evaluations of viral fitness and pathogenicity in animal models in order to understand the mechanism of enhanced transmission and the possible increases in morbidity and mortality rates. Here, we demonstrated that immunity naturally acquired through a prior infection with the first-wave variant does confer nearly complete protection against the B.1.1.7 variant in Syrian hamsters upon reexposure. Strikingly, although the B.1.1.7 variant appears to replicate to a higher level in the nose than the ancestral B.1 variant, it does not induce more severe lung pathology in hamsters.

SARS-CoV-2 B.1.1.7 infection of Syrian hamster does not cause more severe disease and is protected by naturally acquired immunity.

Epidemiological studies have revealed the emergence of multiple SARS-CoV-2 variants of concern (VOC), including the lineage B.1.1.7 that is rapidly replacing old variants. The B.1.1.7 variant has been linked to increased morbidity rates, transmissibility, and potentially mortality (1). To assess viral fitness in vivo and to address whether the B.1.1.7 variant is capable of immune escape, we conducted infection and re-infection studies in naïve and convalescent Syrian hamsters (>10 months old). Hamsters infected by either a B.1.1.7 variant or a B.1 (G614) variant exhibited comparable viral loads and pathology. Convalescent hamsters that were previously infected by the original D614 variant were protected from disease following B.1.1.7 challenge with no observable clinical signs or lung pathology. Altogether, our study did not find that the B.1.1.7 variant significantly differs from the B.1 variant in pathogenicity in hamsters and that natural infection-induced immunity confers protection against a secondary challenge by the B1.1.7 variant.

The PRRA insert at the S1/S2 site modulates cellular tropism of SARS-CoV-2 and ACE2 usage by the closely related Bat RaTG13.

Biochemical and structural analyses suggest that SARS-CoV-2 is well-adapted to infecting humans and the presence of four residues (PRRA) at the S1/S2 site within the spike (S) protein, which may lead to unexpected tissue or host tropism. Here we report that SARS-CoV-2 efficiently utilized ACE2 of 9 species to infect 293T cells. Similarly, pseudoviruses bearing S protein derived from either the bat RaTG13 or pangolin GX, two closely related animal coronaviruses, utilized ACE2 of a diverse range of animal species to gain entry. Removal of PRRA from SARS-CoV-2 S protein displayed distinct effects on pseudoviral entry into different cell types. Unexpectedly, insertion of PRRA into the RaTG13 S protein selectively abrogated the usage of horseshoe bat and pangolin ACE2 but enhanced the usage of mouse ACE2 by the relevant pseudovirus to enter cells. Together, our findings identified a previously unrecognized effect of the PRRA insert on SARS-CoV-2 and RaTG13 S proteins.ImportanceThe four-residue insert (PRRA) at the boundary between the S1and S2 subunits of SARS-CoV-2 has been widely recognized since day 1 for its role in SARS-CoV-2 S protein processing and activation. As this PRRA insert is unique to SARS-CoV-2 among group b betacoronaviruses, it is thought to affect the tissue and species tropism of SARS-CoV-2. We compared the usage of 10 ACE2 orthologs and found that the presence of PRRA not only affects the cellular tropism of SARS-CoV-2 but also modulates the usage of ACE2 orthologs by the closely related bat RaTG13 S protein. The binding of pseudovirions carrying RaTG13 S with a PRRA insert to mouse ACE2 was nearly 2-fold higher than that of pseudovirions carrying RaTG13 S.

Efficacy of recombinant Marek's disease virus vectored vaccines with computationally optimized broadly reactive antigen (COBRA) hemagglutinin insert against genetically diverse H5 high pathogenicity avian influenza viruses.

The genetic and antigenic drift associated with the high pathogenicity avian influenza (HPAI) viruses of Goose/Guangdong (Gs/GD) lineage and the emergence of vaccine-resistant field viruses underscores the need for a broadly protective H5 influenza A vaccine. Here, we tested experimental vector herpesvirus of turkey (vHVT)-H5 vaccines containing either wild-type clade 2.3.4.4A-derived H5 inserts or computationally optimized broadly reactive antigen (COBRA) inserts with challenge by homologous and genetically divergent H5 HPAI Gs/GD lineage viruses in chickens. Direct assessment of protection was confirmed for all the tested constructs, which provided clinical protection against the homologous and heterologous H5 HPAI Gs/GD challenge viruses and significantly decreased oropharyngeal shedding titers compared to the sham vaccine. The cross reactivity was assessed by hemagglutinin inhibition (HI) and focus reduction assay against a panel of phylogenetically and antigenically diverse H5 strains. The COBRA-derived H5 inserts elicited antibody responses against antigenically diverse strains, while the wild-type-derived H5 vaccines elicited protection mostly against close antigenically related clades 2.3.4.4A and 2.3.4.4D viruses. In conclusion, the HVT vector, a widely used replicating vaccine platform in poultry, with H5 insert provides clinical protection and significant reduction of viral shedding against homologous and heterologous challenge. In addition, the COBRA-derived inserts have the potential to be used against antigenically distinct co-circulating viruses and future drift variants.

SARS-CoV-2 infection induces protective immunity and limits transmission in Syrian hamsters.

A critical question in understanding the immunity to SARS-COV-2 is whether recovered patients are protected against re-challenge and transmission upon second exposure. We developed a Syrian hamster model in which intranasal inoculation of just 100 TCID50 virus caused viral pneumonia. Aged hamsters developed more severe disease and even succumbed to SARS-CoV-2 infection, representing the first lethal model using genetically unmodified laboratory animals. After initial viral clearance, the hamsters were re-challenged with 105 TCID50 SARS-CoV-2 and displayed more than 4 log reduction in median viral loads in both nasal washes and lungs in comparison to primary infections. Most importantly, re-challenged hamsters were unable to transmit virus to naïve hamsters, and this was accompanied by the presence of neutralizing antibodies. Altogether, these results show that SARS-CoV-2 infection induces protective immunity that not only prevents re-exposure but also limits transmission in hamsters. These findings may help guide public health policies and vaccine development and aid evaluation of effective vaccines against SARS-CoV-2.

Human COBRA 2 vaccine contains two major epitopes that are responsible for eliciting neutralizing antibody responses against heterologous clades of viruses.

Highly pathogenic H5N1 influenza viruses continue to spread around the globe and reassort with low pathogenic avian influenza viruses often resulting in morbidity and mortality to not only waterfowl, but also poultry. Our group previously developed two hemagglutinin (HA) based vaccines using a methodology termed computationally optimized broadly reactive antigen (COBRA). Each of these HA antigens, Human COBRA 2 (Hu-CO) and Human-Avian COBRA 2 (Hu-Av CO) elicit antibodies with hemagglutination-inhibition (HAI) activity against viruses from various clades, but not always the same viruses. Here, we have sought to identify residues in these two HA molecules that are critical fordifferential HAI activity against various H5Nx influenza viruses. The two HA antigens are remarkedly similar in the globular head region, except for 4 residues at amino acids 140, 141, 155, and 156. By mutating these amino acids in each HA antigen, chimeric HA proteins were used to elicit immune responses in mice. When the Asn-Thr pair at position 155-156 in the Hu-CO HA was converted to the Ser-Ala residues found in the Hu-Av CO HA, the elicited antibodies lost HAI activity against clade 2.3.2.1 H5Nx viruses, such as A/Hubei/01/2010. When this Asn-Thr motif was added at these positions in the Hu-Av CO HA molecule, HAI activity in the elicited sera against A/Hubei/01/2010 was significantly increased. We speculate that a putative N-linked glycosylation at this location in the Hu-CO HA antigen is a key driver in the elicitation of antibodies with HAI activity to different locations on wild-type H5 HA molecules resulting in differential neutralization of viral infection and protection in vivo against H5 influenza virus induced disease.

Structural and antigenic characterization of a computationally-optimized H5 hemagglutinin influenza vaccine.

Influenza A virus is a leading cause of death worldwide. Viruses of the H5 subtype have the potential to induce high mortality, and no vaccines are currently available to protect against H5 influenza viruses in the event of an outbreak. Experimental vaccination with one clade 2 virus does not protect against other subclades. The computationally optimized broadly reactive (COBRA) methodology was previously used to generate a H5 hemagglutinin (HA) antigen (COBRA2) that elicited increased serological breadth against multiple clade 2 H5N1 influenza viruses. In this report, we structurally and antigenically characterized the COBRA2 HA antigen. We examined the biochemical characteristics of the COBRA2 protein and determined the protein is correctly cleaved, properly folded into a trimeric structure, and antigenically correct by probing with HA head- and stem-specific monoclonal antibodies (mAbs). We further probed the antigenicity by examining binding of a panel of H5 mouse mAbs to the COBRA2 antigen, as well as several other HA antigens. We determined the X-ray crystal structure of the COBRA2 HA antigen to 2.8 Šand the protein was observed to be in the expected trimeric form. The COBRA2 HA was structurally similar to the naturally occurring H5 HA antigens and suggests the protein folds similar to known HA structures. Overall, our data allow us to formulate a hypothesis on the mechanism of increased breadth due to vaccination with the COBRA2 HA antigen, which is that the protein incorporates antigenic sites from numerous HA antigens, and elicits mAbs with limited breadth, but with diversity in targeted antigenic sites.

A review of H5Nx avian influenza viruses.

Highly pathogenic avian influenza viruses (HPAIVs), originating from the A/goose/Guangdong/1/1996 H5 subtype, naturally circulate in wild-bird populations, particularly waterfowl, and often spill over to infect domestic poultry. Occasionally, humans are infected with HPAVI H5N1 resulting in high mortality, but no sustained human-to-human transmission. In this review, the replication cycle, pathogenicity, evolution, spread, and transmission of HPAIVs of H5Nx subtypes, along with the host immune responses to Highly Pathogenic Avian Influenza Virus (HPAIV) infection and potential vaccination, are discussed. In addition, the potential mechanisms for Highly Pathogenic Avian Influenza Virus (HPAIV) H5 Reassorted Viruses H5N1, H5N2, H5N6, H5N8 (H5Nx) viruses to transmit, infect, and adapt to the human host are reviewed.

Impact of age and pre-existing influenza immune responses in humans receiving split inactivated influenza vaccine on the induction of the breadth of antibodies to influenza A strains.

Most humans have pre-existing immunity to influenza viruses. In this study, volunteers (ages of 18-85 years) were vaccinated with split, inactivated Fluzone™ influenza vaccine in four consecutive influenza seasons from 2013 to 2016 seasons. The impact of repeated vaccination on breadth and durability of antibodies was assessed as a result of vaccine strain changes. Total IgG anti-hemagglutinin (HA) binding antibodies and hemagglutination-inhibition (HAI) activity increased in all age groups against both influenza A HA components in the vaccine post-vaccination (day 21). However, younger subjects maintained seroprotective titers to the vaccine strains, which resulted in higher seroconversion rates in the elderly, since the HAI titers in elderly subjects were more likely to decline prior to the next season. Young subjects had significant HAI activity against historical, as well as contemporary H1 and H3 vaccine strains from the mid-1980s to present. In contrast, elderly subjects had HAI activity to H1 strains from all years, but were more likely to have HAI activity to older strains from 1918-1950s. They also had a more restricted HAI profile against H3 viruses compared to young subjects recognizing H3N2 influenza viruses from the mid-2000s to present. Vaccine recipients were then categorized by whether subjects seroconverted from a seronegative or seropositive pre-vaccination state. Regardless of age, immunological recall or 'back-boosting' to antigenically related strains were associated with seroconversion to the vaccine strain. Overall, both younger and older people have the ability to mount a breadth of immune responses following influenza vaccination. This report describes how imprinting exposure differs across age groups, influences antibody cross-reactivity to past hemagglutinin antigenic variants, and shapes immune responses elicited by current split inactivated influenza vaccines. Understanding how current influenza vaccines are influenced by pre-existing immunity in people of different ages is critical for designing the next-generation of 'universal' or broadly-protective influenza vaccines.

Traumatic Brain Injury Causes Endothelial Dysfunction in the Systemic Microcirculation through Arginase-1-Dependent Uncoupling of Endothelial Nitric Oxide Synthase.

Endothelial dysfunction is a hallmark of many chronic diseases, including diabetes and long-term hypertension. We show that acute traumatic brain injury (TBI) leads to endothelial dysfunction in rat mesenteric arteries. Endothelial-dependent dilation was greatly diminished 24 h after TBI because of impaired nitric oxide (NO) production. The activity of arginase, which competes with endothelial NO synthase (eNOS) for the common substrate l-arginine, were also significantly increased in arteries, suggesting that arginase-mediated depletion of l-arginine underlies diminished NO production. Consistent with this, substrate restoration by exogenous application of l-arginine or inhibition of arginase recovered endothelial function. Moreover, evidence for increased reactive oxygen species production, a consequence of l-arginine starvation-dependent eNOS uncoupling, was detected in endothelium and plasma. Collectively, our findings demonstrate endothelial dysfunction in a remote vascular bed after TBI, manifesting as impaired endothelial-dependent vasodilation, with increased arginase activity, decreased generation of NO, and increased O2- production. We conclude that blood vessels have a "molecular memory" of neurotrauma, 24 h after injury, because of functional changes in vascular endothelial cells; these effects are pertinent to understanding the systemic inflammatory response that occurs after TBI even in the absence of polytrauma.