Immunogenicity and protective efficacy of a co-formulated two-in-one inactivated whole virus particle COVID-19/influenza vaccine.

Due to the synchronous circulation of seasonal influenza viruses and severe acute respiratory coronavirus 2 (SARS-CoV-2) which causes coronavirus disease 2019 (COVID-19), there is need for routine vaccination for both COVID-19 and influenza to reduce disease severity. Here, we prepared individual WPVs composed of formalin-inactivated SARS-CoV-2 WK 521 (Ancestral strain; Co WPV) or influenza virus [A/California/07/2009 (X-179A) (H1N1) pdm; Flu WPV] to produce a two-in-one Co/Flu WPV. Serum analysis from vaccinated mice revealed that a single dose of Co/Flu WPV induced antigen-specific neutralizing antibodies against both viruses, similar to those induced by either type of WPV alone. Following infection with either virus, mice vaccinated with Co/Flu WPV showed no weight loss, reduced pneumonia and viral titers in the lung, and lower gene expression of proinflammatory cytokines, as observed with individual WPV-vaccinated. Furthermore, a pentavalent vaccine (Co/qFlu WPV) comprising of Co WPV and quadrivalent influenza vaccine (qFlu WPV) was immunogenic and protected animals from severe COVID-19. These results suggest that a single dose of the two-in-one WPV provides efficient protection against SARS-CoV-2 and influenza virus infections with no evidence of vaccine interference in mice. We propose that concomitant vaccination with the two-in-one WPV can be useful for controlling both diseases.

Extraction of the CDRH3 sequence of the mouse antibody repertoire selected upon influenza virus infection by subtraction of the background antibody repertoire.

Historically, antibody reactivity to pathogens and vaccine antigens has been evaluated using serological measurements of antigen-specific antibodies. However, it is difficult to evaluate all antibodies that contribute to various functions in a single assay, such as the measurement of the neutralizing antibody titer. Bulk antibody repertoire analysis using next-generation sequencing is a comprehensive method for analyzing the overall antibody response; however, it is unreliable for estimating antigen-specific antibodies due to individual variation. To address this issue, we propose a method to subtract the background signal from the repertoire of data of interest. In this study, we analyzed changes in antibody diversity and inferred the heavy-chain complementarity-determining region 3 (CDRH3) sequences of antibody clones that were selected upon influenza virus infection in a mouse model using bulk repertoire analysis. A decrease in the diversity of the antibody repertoire was observed upon viral infection, along with an increase in neutralizing antibody titers. Using kernel density estimation of sequences in a high-dimensional sequence space with background signal subtraction, we identified several clusters of CDRH3 sequences induced upon influenza virus infection. Most of these repertoires were detected more frequently in infected mice than in uninfected control mice, suggesting that infection-specific antibody sequences can be extracted using this method. Such an accurate extraction of antigen- or infection-specific repertoire information will be a useful tool for vaccine evaluation in the future. IMPORTANCE: As specific interactions between antigens and cell-surface antibodies trigger the proliferation of B-cell clones, the frequency of each antibody sequence in the samples reflects the size of each clonal population. Nevertheless, it is extremely difficult to extract antigen-specific antibody sequences from the comprehensive bulk antibody sequences obtained from blood samples due to repertoire bias influenced by exposure to dietary antigens and other infectious agents. This issue can be addressed by subtracting the background noise from the post-immunization or post-infection repertoire data. In the present study, we propose a method to quantify repertoire data from comprehensive repertoire data. This method allowed subtraction of the background repertoire, resulting in more accurate extraction of expanded antibody repertoires upon influenza virus infection. This accurate extraction of antigen- or infection-specific repertoire information is a useful tool for vaccine evaluation.

PGL-III, a Rare Intermediate of Mycobacterium leprae Phenolic Glycolipid Biosynthesis, Is a Potent Mincle Ligand.

Although leprosy (Hansen's disease) is one of the oldest known diseases, the pathogenicity of Mycobacterium leprae (M. leprae) remains enigmatic. Indeed, the cell wall components responsible for the immune response against M. leprae are as yet largely unidentified. We reveal here phenolic glycolipid-III (PGL-III) as an M. leprae-specific ligand for the immune receptor Mincle. PGL-III is a scarcely present trisaccharide intermediate in the biosynthetic pathway to PGL-I, an abundant and characteristic M. leprae glycolipid. Using activity-based purification, we identified PGL-III as a Mincle ligand that is more potent than the well-known M. tuberculosis trehalose dimycolate. The cocrystal structure of Mincle and a synthetic PGL-III analogue revealed a unique recognition mode, implying that it can engage multiple Mincle molecules. In Mincle-deficient mice infected with M. leprae, increased bacterial burden with gross pathologies were observed. These results show that PGL-III is a noncanonical ligand recognized by Mincle, triggering protective immunity.

Two Modes of Th1 Polarization Induced by Dendritic-Cell-Priming Adjuvant in Vaccination.

Viral infections are usually accompanied by systemic cytokinemia. Vaccines need not necessarily mimic infection by inducing cytokinemia, but must induce antiviral-acquired immunity. Virus-derived nucleic acids are potential immune-enhancers and particularly good candidates as adjuvants in vaccines in mouse models. The most important nucleic-acid-sensing process involves the dendritic cell (DC) Toll-like receptor (TLR), which participates in the pattern recognition of foreign DNA/RNA structures. Human CD141+ DCs preferentially express TLR3 in endosomes and recognize double-stranded RNA. Antigen cross-presentation occurs preferentially in this subset of DCs (cDCs) via the TLR3-TICAM-1-IRF3 axis. Another subset, plasmacytoid DCs (pDCs), specifically expresses TLR7/9 in endosomes. They then recruit the MyD88 adaptor, and potently induce type I interferon (IFN-I) and proinflammatory cytokines to eliminate the virus. Notably, this inflammation leads to the secondary activation of antigen-presenting cDCs. Hence, the activation of cDCs via nucleic acids involves two modes: (i) with bystander effect of inflammation and (ii) without inflammation. In either case, the acquired immune response finally occurs with Th1 polarity. The level of inflammation and adverse events depend on the TLR repertoire and the mode of response to their agonists in the relevant DC subsets, and could be predicted by assessing the levels of cytokines/chemokines and T cell proliferation in vaccinated subjects. The main differences in the mode of vaccine sought in infectious diseases and cancer are defined by whether it is prophylactic or therapeutic, whether it can deliver sufficient antigens to cDCs, and how it behaves in the microenvironment of the lesion. Adjuvant can be selected on a case-to-case basis.

Inactivated Whole Virus Particle Influenza Vaccine Induces Anti-Neuraminidase Antibodies That May Contribute to Cross-Protection against Heterologous Virus Infection.

Despite the use of vaccines, seasonal influenza remains a risk to public health. We previously proposed the inactivated whole virus particle vaccine (WPV) as an alternative to the widely used split vaccine (SV) for the control of seasonal and pandemic influenza based on the superior priming potency of WPV to that of SV. In this study, we further examined and compared the immunological potency of monovalent WPV and SV of A/California/7/2009 (X-179A) (H1N1) pdm09 (CA/09) to generate immune responses against heterologous viruses, A/Singapore/GP1908/2015 (IVR-180) (H1N1) pdm09 (SG/15), and A/duck/Hokkaido/Vac-3/2007 (H5N1) (DH/07) in mice. Following challenge with a lethal dose of heterologous SG/15, lower virus titer in the lungs and milder weight loss were observed in WPV-vaccinated mice than in SV-vaccinated ones. To investigate the factors responsible for the differences in the protective effect against SG/15, the sera of vaccinated mice were analyzed by hemagglutination-inhibition (HI) and neuraminidase-inhibition (NI) assays to evaluate the antibodies induced against viral hemagglutinin (HA) and neuraminidase (NA), respectively. While the two vaccines induced similar levels of HI antibodies against SG/15 after the second vaccination, only WPV-vaccinated mice induced significantly higher titers of NI antibodies against the strain. Furthermore, given the significant elevation of NI antibody titers against DH/07, an H5N1 avian influenza virus, WPV was also demonstrated to induce NA-inhibiting antibodies that recognize NA of divergent strains. This could be explained by the higher conservation of epitopes of NA among strains than for HA. Taking these findings together, NA-specific antibodies induced by WPV may have contributed to better protection from infection with heterologous influenza virus SG/15, compared with SV. The present results indicate that WPV is an effective vaccine for inducing antibodies against both HA and NA of heterologous viruses and may be a useful vaccine to conquer vaccine strain mismatch.

Point mutation in the stop codon of MAV_RS14660 increases the growth rate of Mycobacterium avium subspecies hominissuis.

Mycobacterium avium subspecies hominissuis (MAH) is a pathogen that causes various non-tuberculous mycobacterial diseases in humans and animals worldwide. Among the genus, MAH is characterized by relatively slow growth. Here, we isolated a rapidly growing variant of the MAH 104 strain. The variant strain (named N104) exhibited an enhanced growth rate and higher motility compared to the parent MAH 104 strain (P104). Whole-genome sequencing analysis of N104 revealed the loss of the stop codon of MAV_RS14660 due to a single nucleotide replacement, resulting in the substitution of the codon for tryptophan. Notably, exclusion of the stop codon ligated the open reading frames and caused the fusion of two adjacent proteins. A revertant parent strain, in which a mutation was introduced to restore the stop codon, revealed that elimination of the stop codon in MAV_RS14660 was responsible for the N104 phenotype. Furthermore, we analysed the phenotypes of the parent and mutated strains by determining the functions of the MAV_RS14660 and MAV_RS14655 coding regions flanking the stop codon. The mutant strains, expected to express a fusion protein, exhibited increased resistance to antimicrobial drugs and exogenous copper toxicity compared to that of the parent strains. These findings suggest that the fusion of the MAV_RS14660- and MAV_RS14655-encoding regions in the mutant N104 strain could be related to the modified functions of these intrinsic proteins.