SARS-CoV-2 Specific Nanobodies Neutralize Different Variants of Concern and Reduce Virus Load in the Brain of h-ACE2 Transgenic Mice.

Since the beginning of the COVID-19 pandemic, there has been a significant need to develop antivirals and vaccines to combat the disease. In this work, we developed llama-derived nanobodies (Nbs) directed against the receptor binding domain (RBD) and other domains of the Spike (S) protein of SARS-CoV-2. Most of the Nbs with neutralizing properties were directed to RBD and were able to block S-2P/ACE2 interaction. Three neutralizing Nbs recognized the N-terminal domain (NTD) of the S-2P protein. Intranasal administration of Nbs induced protection ranging from 40% to 80% after challenge with the WA1/2020 strain in k18-hACE2 transgenic mice. Interestingly, protection was associated with a significant reduction in virus replication in nasal turbinates and a reduction in virus load in the brain. Employing pseudovirus neutralization assays, we identified Nbs with neutralizing capacity against the Alpha, Beta, Delta, and Omicron variants, including a Nb capable of neutralizing all variants tested. Furthermore, cocktails of different Nbs performed better than individual Nbs at neutralizing two Omicron variants (B.1.529 and BA.2). Altogether, the data suggest the potential of SARS-CoV-2 specific Nbs for intranasal treatment of COVID-19 encephalitis.

Exploring the immunogenicity of an insect-specific virus vectored Zika vaccine candidate.

Zika virus (ZIKV) is an important re-emerging flavivirus that presents a significant threat to human health worldwide. Despite its importance, no vaccines are approved for use in humans. Insect-specific flaviviruses (ISFVs) have recently garnered attention as an antigen presentation platform for vaccine development and diagnostic applications. Here, we further explore the safety, immunogenicity, and efficacy of a chimeric ISFV-Zika vaccine candidate, designated Aripo-Zika (ARPV/ZIKV). Our results show a near-linear relationship between increased dose and immunogenicity, with 1011 genome copies (i.e., 108 focus forming units) being the minimum dose required for protection from ZIKV-induced morbidity and mortality in mice. Including boosters did not significantly increase the short-term efficacy of ARPV/ZIKV-vaccinated mice. We also show that weanling mice derived from ARPV/ZIKV-vaccinated dams were completely protected from ZIKV-induced morbidity and mortality upon challenge, suggesting efficient transfer of maternally-derived protective antibodies. Finally, in vitro coinfection studies of ZIKV with Aripo virus (ARPV) and ARPV/ZIKV in African green monkey kidney cells (i.e., Vero-76) showed that ARPV and ARPV/ZIKV remain incapable of replication in vertebrate cells, despite the presence of active ZIKV replication. Altogether, our data continue to support ISFV-based vaccines, and specifically the ARPV backbone is a safe, immunogenic and effective vaccine strategy for flaviviruses.

A bioengineered pseudovirus nanoparticle displaying SARS-CoV 2 RBD fully protects mice from mortality and weight loss caused by SARS-CoV 2 challenge.

The COVID-19 pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused considerable morbidity and mortality worldwide. Although authorized COVID-19 vaccines have been shown highly effective, their significantly lower efficacy against heterologous variants, and the rapid decrease of vaccine-elicited immunity raises serious concerns, calling for improved vaccine tactics. To this end, a pseudovirus nanoparticle (PVNP) displaying the receptor binding domains (RBDs) of SARS-CoV-2 spike, named S-RBD, was generated and shown it as a promising COVID-19 vaccine candidate. The S-RBD PVNP was produced using both prokaryotic and eukaryotic systems. A 3D structural model of the S-RBD PVNPs was built based on the known structures of the S60 particle and RBDs, revealing an S60 particle-based icosahedral symmetry with multiple surface-displayed RBDs that retain authentic conformations and receptor-binding functions. The PVNP is highly immunogenic, eliciting high titers of RBD-specific IgG and neutralizing antibodies in mice. The S-RBD PVNP demonstrated exceptional protective efficacy, and fully (100%) protected K18-hACE2 mice from mortality and weight loss after a lethal SARS-CoV-2 challenge, supporting the S-RBD PVNPs as a potent COVID-19 vaccine candidate. By contrast, a PVNP displaying the N-terminal domain (NTD) of SARS-CoV-2 spike exhibited only 50% protective efficacy. Since the RBD antigens of our PVNP vaccine are adjustable as needed to address the emergence of future variants, and various S-RBD PVNPs can be combined as a cocktail vaccine for broad efficacy, these non-replicating PVNPs offer a flexible platform for a safe, effective COVID-19 vaccine with minimal manufacturing cost and time.

Nanobodies against SARS-CoV-2 reduced virus load in the brain of challenged mice and neutralized Wuhan, Delta and Omicron Variants.

In this work, we developed llama-derived nanobodies (Nbs) directed to the receptor binding domain (RBD) and other domains of the Spike (S) protein of SARS-CoV-2. Nanobodies were selected after the biopanning of two VHH-libraries, one of which was generated after the immunization of a llama (lama glama) with the bovine coronavirus (BCoV) Mebus, and another with the full-length pre-fused locked S protein (S-2P) and the RBD from the SARS-CoV-2 Wuhan strain (WT). Most of the neutralizing Nbs selected with either RBD or S-2P from SARS-CoV-2 were directed to RBD and were able to block S-2P/ACE2 interaction. Three Nbs recognized the N-terminal domain (NTD) of the S-2P protein as measured by competition with biliverdin, while some non-neutralizing Nbs recognize epitopes in the S2 domain. One Nb from the BCoV immune library was directed to RBD but was non-neutralizing. Intranasal administration of Nbs induced protection ranging from 40% to 80% against COVID-19 death in k18-hACE2 mice challenged with the WT strain. Interestingly, protection was not only associated with a significant reduction of virus replication in nasal turbinates and lungs, but also with a reduction of virus load in the brain. Employing pseudovirus neutralization assays, we were able to identify Nbs with neutralizing capacity against the Alpha, Beta, Delta and Omicron variants. Furthermore, cocktails of different Nbs performed better than individual Nbs to neutralize two Omicron variants (B.1.529 and BA.2). Altogether, the data suggest these Nbs can potentially be used as a cocktail for intranasal treatment to prevent or treat COVID-19 encephalitis, or modified for prophylactic administration to fight this disease.

Phylogenetic characterization of Orthobunyaviruses isolated from Trinidad shows evidence of natural reassortment.

The genus Orthobunyavirus is a diverse group of viruses in the family Peribunyaviridae, recently classified into 20 serogroups, and 103 virus species. Although most viruses within these serogroups are phylogenetically distinct, the absence of complete genome sequences has left several viruses incompletely characterized. Here we report the complete genome sequences for 11 orthobunyaviruses isolated from Trinidad, French Guiana, Guatemala, and Panama that were serologically classified into six serogroups and 10 species. Phylogenetic analyses of these 11 newly derived sequences indicate that viruses belonging to the Patois, Capim, Guama, and Group C serocomplexes all have a close genetic origin. We show that three of the 11 orthobunyaviruses characterized (belonging to the Group C and Bunyamwera serogroups) have evidence of histories of natural reassortment through the M genome segment. Our data also suggests that two distinct lineages of Group C viruses concurrently circulate in Trinidad and are transmitted by the same mosquito vectors. This study also highlights the importance of complementing serological identification with nucleotide sequencing when characterizing orthobunyaviruses.

Novel murine models for studying Cache Valley virus pathogenesis and in utero transmission.

Cache Valley virus (CVV) is a prevalent emerging pathogen of significant importance to agricultural and human health in North America. Emergence in livestock can result in substantial agroeconomic losses resulting from the severe embryonic lethality associated with infection during pregnancy. Although CVV pathogenesis has been well described in ruminants, small animal models are still unavailable, which limits our ability to study its pathogenesis and perform preclinical testing of therapeutics. Herein, we explored CVV pathogenesis, tissue tropism, and disease outcomes in a variety of murine models, including immune -competent and -compromised animals. Our results show that development of CVV disease in mice is dependent on innate immune responses, and type I interferon signalling is essential for preventing infection in mice. IFN-αßR-/- mice infected with CVV present with significant disease and lethal infections, with minimal differences in age-dependent pathogenesis, suggesting this model is appropriate for pathogenesis-related, and short- and long-term therapeutic studies. We also developed a novel CVV in utero transmission model that showed high rates of transmission, spontaneous abortions, and congenital malformations during infection. CVV infection presents a wide tissue tropism, with significant amplification in liver, spleen, and placenta tissues. Immune-competent mice are generally resistant to infection, and only show disease in an age dependent manner. Given the high seropositivity rates in regions of North America, and the continuing geographic expansion of competent mosquito vectors, the risk of epidemic and epizootic emergence of CVV is high, and interventions are needed for this important pathogen.

La Crosse Virus Shows Strain-Specific Differences in Pathogenesis.

La Crosse virus (LACV) is the leading cause of pediatric viral encephalitis in North America, and is an important public health pathogen. Historically, studies involving LACV pathogenesis have focused on lineage I strains, but no former work has explored the pathogenesis between or within lineages. Given the absence of LACV disease in endemic regions where a robust entomological risk exists, we hypothesize that some LACV strains are attenuated and demonstrate reduced neuroinvasiveness. Herein, we compared four viral strains representing all three lineages to determine differences in neurovirulence or neuroinvasiveness using three murine models. A representative strain from lineage I was shown to be the most lethal, causing >50% mortality in each of the three mouse studies. However, other strains only presented excessive mortality (>50%) within the suckling mouse neurovirulence model. Neurovirulence was comparable among strains, but viruses differed in their neuroinvasive capacities. Our studies also showed that viruses within lineage III vary in pathogenesis with contemporaneous strains, showing reduced neuroinvasiveness compared to an ancestral strain from the same U.S. state (i.e., Connecticut). These findings demonstrate that LACV strains differ markedly in pathogenesis, and that strain selection is important for assessing vaccine and therapeutic efficacies.

Extreme mitogenomic divergence between two syntopic specimens of Arremon aurantiirostris (Aves: Emberizidae) in central Panama suggests possible cryptic species.

We report the complete mitochondrial genome of two specimens of Orange-billed Sparrow Arremon aurantiirostris from Colón Province, in central Panama. The two specimens were collected on the same day, and at the same locality; however, they showed substantial divergence (6.3% average pairwise divergence among coding genes). A survey of ND2 sequence variation across Panama suggests that this divergence is the result of geographic differentiation and secondary contact. This high level of mitochondrial divergence among co-occurring individuals raises the possibility of multiple biological species in Orange-billed Sparrows. Our results are yet another demonstration that much remains to be discovered regarding avian biodiversity in Panama and throughout the Neotropics.