Further Characterization of the Antiviral Transmembrane Protein MARCH8.

The cellular transmembrane protein MARCH8 impedes the incorporation of various viral envelope glycoproteins, such as the HIV-1 envelope glycoprotein (Env) and vesicular stomatitis virus G-glycoprotein (VSV-G), into virions by downregulating them from the surface of virus-producing cells. This downregulation significantly reduces the efficiency of virus infection. In this study, we aimed to further characterize this host protein by investigating its species specificity and the domains responsible for its antiviral activity, as well as its ability to inhibit cell-to-cell HIV-1 infection. We found that the antiviral function of MARCH8 is well conserved in the rhesus macaque, mouse, and bovine versions. The RING-CH domains of these versions are functionally important for inhibiting HIV-1 Env and VSV-G-pseudovirus infection, whereas tyrosine motifs are crucial for the former only, consistent with findings in human MARCH8. Through analysis of chimeric proteins between MARCH8 and non-antiviral MARCH3, we determined that both the N-terminal and C-terminal cytoplasmic tails, as well as presumably the N-terminal transmembrane domain, of MARCH8 are critical for its antiviral activity. Notably, we found that MARCH8 is unable to block cell-to-cell HIV-1 infection, likely due to its insufficient downregulation of Env. These findings offer further insights into understanding the biology of this antiviral transmembrane protein.

Checkpoint inhibitor-expressing lentiviral vaccine suppresses tumor growth in preclinical cancer models.

BACKGROUND: While immunotherapy has been highly successful for the treatment of some cancers, for others, the immune response to tumor antigens is weak leading to treatment failure. The resistance of tumors to checkpoint inhibitor therapy may be caused by T cell exhaustion resulting from checkpoint activation. METHODS: In this study, lentiviral vectors that expressed T cell epitopes of an experimentally introduced tumor antigen, ovalbumin, or the endogenous tumor antigen, Trp1 were developed. The vectors coexpressed CD40 ligand (CD40L), which served to mature the dendritic cells (DCs), and a soluble programmed cell death protein 1 (PD-1) microbody to prevent checkpoint activation. Vaccination of mice bearing B16.OVA melanomas with vector-transduced DCs induced the proliferation and activation of functional, antigen-specific, cytolytic CD8 T cells. RESULTS: Vaccination induced the expansion of CD8 T cells that infiltrated the tumors to suppress tumor growth. Vector-encoded CD40L and PD-1 microbody increased the extent of tumor growth suppression. Adoptive transfer demonstrated that the effect was mediated by CD8 T cells. Direct injection of the vector, without the need for ex vivo transduction of DCs, was also effective. CONCLUSIONS: This study suggests that therapeutic vaccination that induces tumor antigen-specific CD8 T cells coupled with a vector-expressed checkpoint inhibitor can be an effective means to suppress the growth of tumors that are resistant to conventional immunotherapy.

Two cases of bilateral reverse shoulder arthroplasty performed in patients with rheumatoid arthritis.

Bilateral shoulder joint disorders caused by rheumatoid arthritis significantly impair daily functioning owing to a lack of contralateral compensation. In Japan, reverse shoulder joint prostheses were approved in 2014. This was expected to improve the surgical outcomes of rheumatoid shoulder arthroplasty. We report two patients with rheumatoid arthritis who underwent bilateral reverse shoulder arthroplasty. This study aims to evaluate their postoperative clinical outcomes and activities of daily living. The patients were women in their 70s with stage III class 2 rheumatoid arthritis. Their treatment and postoperative activities of daily living were retrospectively reviewed. The first patient underwent the inlay type and experienced a residual limitation of external rotation postoperatively; therefore, she was restricted to dress with front-open clothes. However, she was able to undress after the lining of the garment was changed to a slippery material. The second patient underwent the onlay type and showed almost no limitations in postoperative activities of daily living. She was able to undress with an external rotation of 40-50°. Bilateral reverse shoulder arthroplasty improved range of motion, the Japanese Orthopaedic Association shoulder score, and functional outcomes. Only a few difficulties were encountered in the activities of daily living.

Restriction of SARS-CoV-2 replication by receptor transporter protein 4 (RTP4).

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is subject to restriction by several interferon-inducible host proteins. To identify novel factors that limit replication of the virus, we tested a panel of genes that we found were induced by interferon treatment of primary human monocytes by RNA sequencing. Further analysis showed that one of the several candidates genes tested, receptor transporter protein 4 (RTP4), that had previously been shown to restrict flavivirus replication, prevented the replication of the human coronavirus HCoV-OC43. Human RTP4 blocked the replication of SARS-CoV-2 in susceptible ACE2.CHME3 cells and was active against SARS-CoV-2 Omicron variants. The protein prevented the synthesis of viral RNA, resulting in the absence of detectable viral protein synthesis. RTP4 bound the viral genomic RNA and the binding was dependent on the conserved zinc fingers in the amino-terminal domain. Expression of the protein was strongly induced in SARS-CoV-2-infected mice although the mouse homolog was inactive against the virus, suggesting that the protein is active against another virus that remains to be identified. IMPORTANCE The rapid spread of a pathogen of human coronavirus (HCoV) family member, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), around the world has led to a coronavirus disease 2019 (COVID-19) pandemic. The COVID-19 pandemic spread highlights the need for rapid identification of new broad-spectrum anti-coronavirus drugs and screening of antiviral host factors capable of inhibiting coronavirus infection. In the present work, we identify and characterize receptor transporter protein 4 (RTP4) as a host restriction factor that restricts coronavirus infection. We examined the antiviral role of hRTP4 toward the coronavirus family members including HCoV-OC43, SARS-CoV-2, Omicron BA.1, and BA.2. Molecular and biochemical analysis showed that hRTP4 binds to the viral RNA and targets the replication phase of viral infection and is associated with reduction of nucleocapsid protein. Significant higher levels of ISGs were observed in SARS-CoV-2 mouse model, suggesting the role of RTP4 in innate immune regulation in coronavirus infection. The identification of RTP4 reveals a potential target for therapy against coronavirus infection.

Vectored immunoprophylaxis and treatment of SARS-CoV-2 infection in a preclinical model.

Vectored immunoprophylaxis was first developed as a means of establishing engineered immunity to HIV using an adenoassociated viral vector expressing a broadly neutralizing antibody. We applied this concept to establish long-term prophylaxis against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in a mouse model using adenoassociated virus and lentiviral vectors expressing a high-affinity angiotensin-converting enzyme 2 (ACE2) decoy. Administration of decoy-expressing (adenoassociated virus) AAV2.retro and AAV6.2 vectors by intranasal instillation or intramuscular injection protected mice against high-titered SARS-CoV-2 infection. AAV and lentiviral vectored immunoprophylaxis was durable and was active against SARS-CoV-2 Omicron subvariants. The AAV vectors were also effective therapeutically when administered postinfection. Vectored immunoprophylaxis could be of value for immunocompromised individuals for whom vaccination is not practical and as a means to rapidly establish protection from infection. Unlike monoclonal antibody therapy, the approach is expected to remain active despite continued evolution viral variants.

Single-epitope T cell-based vaccine protects against SARS-CoV-2 infection in a preclinical animal model.

Currently authorized COVID-19 vaccines induce humoral and cellular responses to epitopes in the SARS-CoV-2 spike protein, though the relative roles of antibodies and T cells in protection are not well understood. To understand the role of vaccine-elicited T cell responses in protection, we established a T cell-only vaccine using a DC-targeted lentiviral vector expressing single CD8+ T cell epitopes of the viral nucleocapsid, spike, and ORF1. Immunization of angiotensin-converting enzyme 2-transgenic mice with ex vivo lentiviral vector-transduced DCs or by direct injection of the vector induced the proliferation of functional antigen-specific CD8+ T cells, resulting in a 3-log decrease in virus load upon live virus challenge that was effective against the ancestral virus and Omicron variants. The Pfizer/BNT162b2 vaccine was also protective in mice, but the antibodies elicited did not cross-react on the Omicron variants, suggesting that the protection was mediated by T cells. The studies suggest that the T cell response plays an important role in vaccine protection. The findings suggest that the incorporation of additional T cell epitopes into current vaccines would increase their effectiveness and broaden protection.

Delta-Omicron recombinant escapes therapeutic antibody neutralization.

The emergence of recombinant viruses is a threat to public health, as recombination may integrate variant-specific features that together result in escape from treatment or immunity. The selective advantages of recombinant SARS-CoV-2 isolates over their parental lineages remain unknown. We identified a Delta-Omicron (AY.45-BA.1) recombinant in an immunosuppressed transplant recipient treated with monoclonal antibody Sotrovimab. The single recombination breakpoint is located in the spike N-terminal domain adjacent to the Sotrovimab binding site. While Delta and BA.1 are sensitive to Sotrovimab neutralization, the Delta-Omicron recombinant is highly resistant. To our knowledge, this is the first described instance of recombination between circulating SARS-CoV-2 variants as a functional mechanism of resistance to treatment and immune escape.

Prophylaxis and treatment of SARS-CoV-2 infection by an ACE2 receptor decoy in a preclinical animal model.

The emergence of SARS-CoV-2 variants with highly mutated spike proteins has presented an obstacle to the use of monoclonal antibodies for the prevention and treatment of SARS-CoV-2 infection. We show that a high-affinity receptor decoy protein in which a modified ACE2 ectodomain is fused to a single domain of an immunoglobulin heavy chain Fc region dramatically suppressed virus loads in mice upon challenge with a high dose of parental SARS-CoV-2 or Omicron variants. The decoy also potently suppressed virus replication when administered shortly post-infection. The decoy approach offers protection against the current viral variants and, potentially, against SARS-CoV-2 variants that may emerge with the continued evolution of the spike protein or novel viruses that use ACE2 for virus entry.

Prophylaxis and Treatment of SARS-CoV-2 infection by an ACE2 Receptor Decoy.

The emergence of SARS-CoV-2 variants with highly mutated spike proteins has presented an obstacle to the use of monoclonal antibodies for the prevention and treatment of SARS-CoV-2 infection. We show that a high affinity receptor decoy protein in which a modified ACE2 ectodomain is fused to a single domain of an immunoglobulin heavy chain Fc region dramatically suppressed virus loads in mice upon challenge with a high dose of parental SARS-CoV-2 or Omicron variants. The decoy also potently suppressed virus replication when administered shortly post-infection. The decoy approach offers protection against the current viral variants and, potentially, against SARS-CoV-2 variants that may emerge with the continued evolution of the spike protein or novel viruses that use ACE2 for virus entry.

Vectored Immunoprophylaxis and Treatment of SARS-CoV-2 Infection.

Vectored immunoprophylaxis was first developed as a means to establish engineered immunity to HIV through the use of an adeno-associated viral vector expressing a broadly neutralizing antibody. We have applied this concept to establish long-term prophylaxis against SARS-CoV-2 by adeno-associated and lentiviral vectors expressing a high affinity ACE2 decoy receptor. Administration of decoy-expressing AAV vectors based on AAV2.retro and AAV6.2 by intranasal instillation or intramuscular injection protected mice against high-titered SARS-CoV-2 infection. AAV and lentiviral vectored immunoprophylaxis was durable and active against recent SARS-CoV-2 Omicron subvariants. The AAV vectors were also effective when administered up to 24 hours post-infection. Vectored immunoprophylaxis could be of value for immunocompromised individuals for whom vaccination is not practical and as a means to rapidly establish protection from infection. Unlike monoclonal antibody therapy, the approach is expected to remain active despite continued evolution viral variants.

Single-virus tracking reveals variant SARS-CoV-2 spike proteins induce ACE2-independent membrane interactions.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) became a global health crisis after its emergence in 2019. Replication of the virus is initiated by binding of the viral spike (S) protein to human angiotensin-converting enzyme 2 (ACE2) on the target cell surface. Mutations acquired by SARS-CoV-2 S variants likely influence virus-target cell interaction. Here, using single-virus tracking to capture these initial steps, we observe how viruses carrying variant S interact with target cells. Specificity for ACE2 occurs for viruses with the reference sequence or D614G mutation. Analysis of the Alpha, Beta, and Delta SARS-CoV-2 variant S proteins revealed a progressive altered cell interaction with a reduced dependence on ACE2. Notably, the Delta variant S affinity was independent of ACE2. These enhanced interactions may account for the increased transmissibility of variants. Knowledge of how mutations influence cell interaction is essential for vaccine development against emerging variants of SARS-CoV-2.

Directly injected lentiviral vector-based T cell vaccine protects mice against acute and chronic viral infection.

Lentiviral vector-based dendritic cell vaccines induce protective T cell responses against viral infection and cancer in animal models. In this study, we tested whether preventative and therapeutic vaccination could be achieved by direct injection of antigen-expressing lentiviral vector, obviating the need for ex vivo transduction of dendritic cells. Injected lentiviral vector preferentially transduced splenic dendritic cells and resulted in long-term expression. Injection of a lentiviral vector encoding an MHC class I-restricted T cell epitope of lymphocytic choriomeningitis virus (LCMV) and CD40 ligand induced an antigen-specific cytolytic CD8+ T lymphocyte response that protected the mice from infection. The injection of chronically infected mice with a lentiviral vector encoding LCMV MHC class I and II T cell epitopes and a soluble programmed cell death 1 microbody rapidly cleared the virus. Vaccination by direct injection of lentiviral vector was more effective in sterile alpha motif and HD-domain containing protein 1-knockout (SAMHD1-knockout) mice, suggesting that lentiviral vectors containing Vpx, a lentiviral protein that increases the efficiency of dendritic cell transduction by inducing the degradation of SAMHD1, would be an effective strategy for the treatment of chronic disease in humans.

Engineered multivalent self-assembled binder protein against SARS-CoV-2 RBD.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused a global pandemic since December 2019, and with it, a push for innovations in rapid testing and neutralizing antibody treatments in an effort to solve the spread and fatality of the disease. One such solution to both of these prevailing issues is targeting the interaction of SARS-CoV-2 spike receptor binding domain (RBD) with the human angiotensin-converting enzyme 2 (ACE2) receptor protein. Structural studies have shown that the N-terminal alpha-helix comprised of the first 23 residues of ACE2 plays an important role in this interaction. Where it is typical to design a binding domain to fit a target, we have engineered a protein that relies on multivalency rather than the sensitivity of a monomeric ligand to provide avidity to its target by fusing the N-terminal helix of ACE2 to the coiled-coil domain of the cartilage oligomeric matrix protein. The resulting ACE-MAP is able to bind to the SARS-CoV-2 RBD with improved binding affinity, is expressible in E. coli, and is thermally stable and relatively small (62 kDa). These properties suggest ACE-MAP and the MAP scaffold to be a promising route towards developing future diagnostics and therapeutics to SARS-CoV-2.

Efficacy of peficitinib in two patients with rheumatoid arthritis on maintenance hemodialysis.

Objective: Treatment options for patients with rheumatoid arthritis on maintenance hemodialysis with an inadequate response to biologic agents have not been reported. In this report, we describe two patients who achieved remission after treatment with peficitinib. Methods: Two 69- and 85-year-old patients with rheumatoid arthritis on maintenance hemodialysis were previously treated with biologics and started on peficitinib 100 mg/day after the secondary failure of biologics. Discussion: In the two cases presented here, rheumatoid arthritis was almost in remission and there were no adverse events, although the patients were switched to peficitinib after secondary failure of the biologic agents. Among Janus kinase inhibitors, peficitinib has the lowest renal excretion; therefore, its administration in patients on dialysis is not contraindicated according to the package insert in Japan. The use of biologic agents in patients on hemodialysis has been reported to be associated with a high incidence of infections; therefore, care should be taken to avoid infections when administering Janus kinase inhibitors. Conclusion: Janus kinase inhibitors with low renal excretion, such as peficitinib, may be effective in patients with rheumatoid arthritis on maintenance hemodialysis who have an inadequate response to biologic agents.

Resistance of SARS-CoV-2 Omicron BA.1 and BA.2 Variants to Vaccine-Elicited Sera and Therapeutic Monoclonal Antibodies.

The recent emergence of the Omicron BA.1 and BA.2 variants with heavily mutated spike proteins has posed a challenge to the effectiveness of current vaccines and to monoclonal antibody therapy for severe COVID-19. After two immunizations of individuals with no history of previous SARS-CoV-2 infection with BNT162b2 vaccine, neutralizing titer against BA.1 and BA.2 were 20-fold decreased compared to titers against the parental D614G virus. A third immunization boosted overall neutralizing titers by about 5-fold but titers against BA.1 and BA.2 remained about 10-fold below that of D614G. Both Omicron variants were highly resistant to several of the emergency use authorized therapeutic monoclonal antibodies. The variants were highly resistant to Regeneron REGN10933 and REGN10987 and Lilly LY-CoV555 and LY-CoV016 while Vir-7831 and the mixture of AstraZeneca monoclonal antibodies AZD8895 and AZD1061 were significantly decreased in neutralizing titer. Strikingly, a single monoclonal antibody LY-CoV1404 potently neutralized both Omicron variants.

Inflammatory responses in the placenta upon SARS-CoV-2 infection late in pregnancy.

The effect of SARS-CoV-2 infection on placental function is not well understood. Analysis of placentas from women who tested positive at delivery showed SARS-CoV-2 genomic and subgenomic RNA in 22 out of 52 placentas. Placentas from two mothers with symptomatic COVID-19 whose pregnancies resulted in adverse outcomes for the fetuses contained high levels of viral Alpha variant RNA. The RNA was localized to the trophoblasts that cover the fetal chorionic villi in direct contact with maternal blood. The intervillous spaces and villi were infiltrated with maternal macrophages and T cells. Transcriptome analysis showed an increased expression of chemokines and pathways associated with viral infection and inflammation. Infection of placental cultures with live SARS-CoV-2 and spike protein-pseudotyped lentivirus showed infection of syncytiotrophoblast and, in rare cases, endothelial cells mediated by ACE2 and Neuropilin-1. Viruses with Alpha, Beta, and Delta variant spikes infected the placental cultures at significantly greater levels.

Increased resistance of SARS-CoV-2 Omicron variant to neutralization by vaccine-elicited and therapeutic antibodies.

BACKGROUND: SARS-CoV-2 vaccines currently authorized for emergency use have been highly successful in preventing infection and lessening disease severity. The vaccines maintain effectiveness against earlier SARS-CoV-2 Variants of Concern but the heavily mutated, highly transmissible Omicron variant presents an obstacle both to vaccine protection and monoclonal antibody therapies. METHODS: Pseudotyped lentiviruses were incubated with serum from vaccinated and boosted donors or therapeutic monoclonal antibody and then applied to target cells. After 2 days, luciferase activity was measured in a microplate luminometer. Resistance mutations of the Omicron spike were identified using point-mutated spike protein pseudotypes and mapped onto the three-dimensional spike protein structure. FINDINGS: Virus with the Omicron spike protein was 26-fold resistant to neutralization by recovered donor sera and 26-34-fold resistance to Pfizer BNT162b2 and Moderna vaccine-elicited antibodies following two immunizations. A booster immunization increased neutralizing titres against Omicron. Neutralizing titres against Omicron were increased in the sera with a history of prior SARS-CoV-2 infection. Analysis of the therapeutic monoclonal antibodies showed that the Regeneron and Eli Lilly monoclonal antibodies were ineffective against the Omicron pseudotype while Sotrovimab and Evusheld were partially effective. INTERPRETATION: The results highlight the benefit of a booster immunization to protect against the Omicron variant and demonstrate the challenge to monoclonal antibody therapy. The decrease in neutralizing titres against Omicron suggest that much of the vaccine efficacy may rely on T cells. FUNDING: The work was funded by grants from the NIH to N.R.L. (DA046100, AI122390 and AI120898) and 55 to M.J.M. (UM1AI148574).

Delta-Omicron recombinant escapes therapeutic antibody neutralization.

Background: The emergence of recombinant viruses is a threat to public health. Recombination of viral variants may combine variant-specific features that together catalyze viral escape from treatment or immunity. The selective advantages of recombinant SARS-CoV-2 isolates over their parental lineages remain unknown. Methods: Multi-method amplicon and metagenomic sequencing of a clinical swab and the in vitro grown virus allowed for high-confidence detection of a novel recombinant variant. Mutational, phylogeographic, and structural analyses determined features of the recombinant genome and spike protein. Neutralization assays using infectious as well as pseudotyped viruses and point mutants thereof defined the recombinant's sensitivity to a panel of monoclonal antibodies and sera from vaccinated and/or convalescent individuals. Results: A novel Delta-Omicron SARS-CoV-2 recombinant was identified in an unvaccinated, immunosuppressed kidney transplant recipient treated with monoclonal antibody Sotrovimab. The recombination breakpoint is located in the spike N-terminal domain, adjacent to the Sotrovimab quaternary binding site, and results in a 5'-Delta AY.45 and a 3'-Omicron BA.1 mosaic spike protein. Delta and BA.1 are sensitive to Sotrovimab neutralization, whereas the Delta-Omicron recombinant is highly resistant to Sotrovimab, both with and without the RBD resistance mutation E340D. Conclusions: Recombination between circulating SARS-CoV-2 variants can functionally contribute to immune escape. It is critical to validate phenotypes of mosaic viruses and monitor immunosuppressed COVID-19 patients treated with monoclonal antibodies for the selection of recombinant and immune escape variants. (Funded by NYU, the National Institutes of Health, and others).

Neutralization of SARS-CoV-2 Variants by mRNA and Adenoviral Vector Vaccine-Elicited Antibodies.

The increasing prevalence of SARS-CoV-2 variants has raised concerns regarding possible decreases in vaccine effectiveness. Here, neutralizing antibody titers elicited by mRNA-based and adenoviral vector-based vaccines against variant pseudotyped viruses were measured. BNT162b2 and mRNA-1273-elicited antibodies showed modest neutralization resistance against Beta, Delta, Delta plus and Lambda variants whereas Ad26.COV2.S-elicited antibodies from a significant fraction of vaccinated individuals had less neutralizing titer (IC50 <50). The data underscore the importance of surveillance for breakthrough infections that result in severe COVID-19 and suggest a potential benefit by second immunization following Ad26.COV2.S to increase protection from current and future variants.

Variable susceptibility of intestinal organoid-derived monolayers to SARS-CoV-2 infection.

Gastrointestinal effects associated with Coronavirus Disease 2019 (COVID-19) are highly variable for reasons that are not understood. In this study, we used intestinal organoid-derived cultures differentiated from primary human specimens as a model to examine interindividual variability. Infection of intestinal organoids derived from different donors with Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) resulted in orders of magnitude differences in virus replication in small intestinal and colonic organoid-derived monolayers. Susceptibility to infection correlated with angiotensin I converting enzyme 2 (ACE2) expression level and was independent of donor demographic or clinical features. ACE2 transcript levels in cell culture matched the amount of ACE2 in primary tissue, indicating that this feature of the intestinal epithelium is retained in the organoids. Longitudinal transcriptomics of organoid-derived monolayers identified a delayed yet robust interferon signature, the magnitude of which corresponded to the degree of SARS-CoV-2 infection. Interestingly, virus with the Omicron variant spike (S) protein infected the organoids with the highest infectivity, suggesting increased tropism of the virus for intestinal tissue. These results suggest that heterogeneity in SARS-CoV-2 replication in intestinal tissues results from differences in ACE2 levels, which may underlie variable patient outcomes.