Supplementation of seasonal vaccine with multi-subtype neuraminidase and M2 ectodomain virus-like particle improves protection against homologous and heterologous influenza viruses in aged mice.

The conventional inactivated split seasonal influenza vaccine offers low efficacy, particularly in the elderly and against antigenic variants. Here, to improve the efficacy of seasonal vaccination for the elderly population, we tested whether supplementing seasonal bivalent (H1N1 + H3N2) split (S) vaccine with M2 ectodomain repeat and multi-subtype consensus neuraminidase (NA) proteins (N1 NA + N2 NA + flu B NA) on a virus-like particle (NA-M2e) would induce enhanced cross-protection against different influenza viruses in aged mice. Immunization with split vaccine plus NA-M2e (S + NA-M2e) increased vaccine-specific IgG antibodies towards T-helper type 1 responses and hemagglutination inhibition titers. Aged mice with NA-M2e supplemented vaccination were protected against homologous and heterologous viruses at higher efficacies, as evidenced by preventing weight loss, lowering lung viral loads, inducing broadly cross-protective humoral immunity, and IFN-γ+ CD4 and CD8 T cell responses than those with seasonal vaccine. Overall, this study supports a new strategy of NA-M2e supplemented vaccination to enhance protection against homologous and antigenically different viruses in the elderly.

Development of an attenuated potato virus Y mutant carrying multiple mutations in helper-component protease for cross-protection.

Tobacco (Nicotiana tabacum) is one of the major cash crops in China. Potato virus Y (PVY), a representative member of the genus Potyvirus, greatly reduces the quality and yield of tobacco leaves by inducing veinal necrosis. Mild strain-mediated cross-protection is an attractive method of controlling diseases caused by PVY. Currently, there is a lack of effective and stable attenuated PVY mutants. Potyviral helper component-protease (HC-Pro) is a likely target for the development of mild strains. Our previous studies showed that the residues lysine at positions 124 and 182 (K124 and K182) in HC-Pro were involved in PVY virulence, and the conserved KITC motif in HC-Pro was involved in aphid transmission. In this study, to improve the stability of PVY mild strains, K at position 50 (K50) in KITC motif, K124, and K182 were separately substituted with glutamic acid (E), leucine (L), and arginine (R), resulting in a triple-mutant PVY-HCELR. The mutant PVY-HCELR had attenuated virulence and did not induce leaf veinal necrosis symptoms in tobacco plants and could not be transmitted by Myzus persicae. Furthermore, PVY-HCELR mutant was genetically stable after six serial passages, and only caused mild mosaic symptoms in tobacco plants even at 90 days post inoculation. The tobacco plants cross-protected by PVY-HCELR mutant showed high resistance to the wild-type PVY. This study showed that PVY-HCELR mutant was a promising mild mutant for cross-protection to control PVY.

Cross-protection induced by highly conserved human B, CD4+, and CD8+ T-cell epitopes-based vaccine against severe infection, disease, and death caused by multiple SARS-CoV-2 variants of concern.

Background: The coronavirus disease 2019 (COVID-19) pandemic has created one of the largest global health crises in almost a century. Although the current rate of Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infections has decreased significantly, the long-term outlook of COVID-19 remains a serious cause of morbidity and mortality worldwide, with the mortality rate still substantially surpassing even that recorded for influenza viruses. The continued emergence of SARS-CoV-2 variants of concern (VOCs), including multiple heavily mutated Omicron sub-variants, has prolonged the COVID-19 pandemic and underscores the urgent need for a next-generation vaccine that will protect from multiple SARS-CoV-2 VOCs. Methods: We designed a multi-epitope-based coronavirus vaccine that incorporated B, CD4+, and CD8+ T- cell epitopes conserved among all known SARS-CoV-2 VOCs and selectively recognized by CD8+ and CD4+ T-cells from asymptomatic COVID-19 patients irrespective of VOC infection. The safety, immunogenicity, and cross-protective immunity of this pan-variant SARS-CoV-2 vaccine were studied against six VOCs using an innovative triple transgenic h-ACE-2-HLA-A2/DR mouse model. Results: The pan-variant SARS-CoV-2 vaccine (i) is safe , (ii) induces high frequencies of lung-resident functional CD8+ and CD4+ TEM and TRM cells , and (iii) provides robust protection against morbidity and virus replication. COVID-19-related lung pathology and death were caused by six SARS-CoV-2 VOCs: Alpha (B.1.1.7), Beta (B.1.351), Gamma or P1 (B.1.1.28.1), Delta (lineage B.1.617.2), and Omicron (B.1.1.529). Conclusion: A multi-epitope pan-variant SARS-CoV-2 vaccine bearing conserved human B- and T- cell epitopes from structural and non-structural SARS-CoV-2 antigens induced cross-protective immunity that facilitated virus clearance, and reduced morbidity, COVID-19-related lung pathology, and death caused by multiple SARS-CoV-2 VOCs.

Antigenic similarities and clinical cross-protection between variant and classic non-viscous strains of Moritella viscosa in Atlantic salmon in Norway.

Moritella viscosa (M. viscosa) is one of the major etiological agents of winter-ulcers in Atlantic salmon (Salmo salar) in Norway. Outbreaks of ulcerative disease in farmed fish occur across the North Atlantic region, causing reduced animal welfare and economical challenges, and are of hindrance for sustainable growth within the industry. Commercially available multivalent core vaccines containing inactivated bacterin of M. viscosa reduce mortality and clinical signs related to winter ulcer disease. It has previously been described two major genetic clades within M. viscosa, typical (hereafter referred to as classic) and variant, based on gyrB sequencing. In addition, there are phenotypical traits such as viscosity that may differ between different types of isolates. Western blot using salmon plasma showed that classic non-viscous strains are antigenically different from the classic viscous type included in core vaccines. Further, Western blot also showed that there are similarities in binding patterns between Norwegian variant and classic non-viscous isolates, indicating they may be antigenically related. Vaccination-challenge trials using Norwegian gyrB-classic non-viscous isolates of M. viscosa, demonstrate that the isolates from the classic clade that are included in current commercial multivalent core vaccines, provide limited cross protection against the emerging non-viscous strains. However, a vaccine recently approved for marketing authorization in Norway, containing inactivated antigen of a variant M. viscosa strain, demonstrates reduced mortality as well as clinical signs caused by infections with the classic non-viscous M. viscosa isolated from outbreaks in Norwegian salmon farms. The study shows that there are antigenic similarities between variant and classic non-viscous types of M. viscosa, and these similarities are mirrored in the observed cross-protection in vaccination-challenge trials.

Clade 2.3.4.4 H5 chimeric cold-adapted attenuated influenza vaccines induced cross-reactive protection in mice and ferrets.

IMPORTANCE: Clade 2.3.4.4 H5Nx avian influenza viruses (AIVs) have circulated globally and caused substantial economic loss. Increasing numbers of humans have been infected with Clade 2.3.4.4 H5N6 AIVs in recent years. Only a few human influenza vaccines have been licensed to date. However, the licensed live attenuated influenza virus vaccine exhibited the potential of being recombinant with the wild-type influenza A virus (IAV). Therefore, we developed a chimeric cold-adapted attenuated influenza vaccine based on the Clade 2.3.4.4 H5 AIVs. These H5 vaccines demonstrate the advantage of being non-recombinant with circulated IAVs in the future influenza vaccine study. The findings of our current study reveal that these H5 vaccines can induce cross-reactive protective efficacy in mice and ferrets. Our H5 vaccines may provide a novel option for developing human-infected Clade 2.3.4.4 H5 AIV vaccines.

One-step preparation of a self-assembled bioconjugate nanovaccine against Brucella.

Brucellosis, caused by Brucella, is a severe zoonosis, and the current Brucella live attenuated vaccine cannot be used in humans due to major safety risks. Although polysaccharide antigens can be used to prepare the Brucella vaccine, their lower immunogenicity limits them from producing efficient and broad protection. In this study, we produced a high-performance bioconjugate nanovaccine against different species of Brucella by introducing a self-assembly nanoparticle platform and an O-linked glycosylation system into Yersinia enterocolitica serotype O:9, which has an O-polysaccharide composed of the same unit as Brucella. After successfully preparing the vaccine and confirming its stability, we subsequently demonstrated the safety of the vaccine in mice by high-dose immunization. Then, by a series of mouse experiments, we found that the nanovaccine greatly promoted antibody responses. In particular, the increase of IgG2a was more obvious than that of IgG1. Most importantly, this nanovaccine could provide cross-protection against B. abortus, B. melitensis, and B. suis strains by lethal dose challenged models, and could improve the clearance of B. melitensis, the most common pathogenic species in human brucellosis, by non-lethal dose infection. Overall, for the first time, we biocoupled polysaccharide antigens with nano carriers to prepare a Brucella vaccine, which showed pronounced and extensive protective effects in mice. Thus, we provided a potential candidate vaccine and a new direction for Brucella vaccine design.

Single-Component Multilayered Self-Assembling Protein Nanoparticles Displaying Extracellular Domains of Matrix Protein 2 as a Pan-influenza A Vaccine.

The development of a cross-protective pan-influenza A vaccine remains a significant challenge. In this study, we designed and evaluated single-component self-assembling protein nanoparticles (SApNPs) presenting the conserved extracellular domain of matrix protein 2 (M2e) as vaccine candidates against influenza A viruses. The SApNP-based vaccine strategy was first validated for human M2e (hM2e) and then applied to tandem repeats of M2e from human, avian, and swine hosts (M2ex3). Vaccination with M2ex3 displayed on SApNPs demonstrated higher survival rates and less weight loss compared to the soluble M2ex3 antigen against the lethal challenges of H1N1 and H3N2 in mice. M2ex3 I3-01v9a SApNPs formulated with a squalene-based adjuvant were retained in the lymph node follicles over 8 weeks and induced long-lived germinal center reactions. Notably, a single low dose of M2ex3 I3-01v9a SApNP formulated with a potent adjuvant, either a Toll-like receptor 9 (TLR9) agonist or a stimulator of interferon genes (STING) agonist, conferred 90% protection against a lethal H1N1 challenge in mice. With the ability to induce robust and durable M2e-specific functional antibody and T cell responses, the M2ex3-presenting I3-01v9a SApNP provides a promising pan-influenza A vaccine candidate.

Immune responses to Neisseria gonorrhoeae and implications for vaccine development.

Neisseria gonorrheoae is the causative agent of gonorrhea, a sexually transmitted infection responsible for a major burden of disease with a high global prevalence. Protective immunity to infection is often not observed in humans, possible due to high variability of key antigens, induction of blocking antibodies, or a large number of infections being relatively superficial and not inducing a strong immune response. N. gonorrhoeae is a strictly human pathogen, however, studies using mouse models provide useful insights into the immune response to gonorrhea. In mice, N. gonorrhoea appears to avoid a protective Th1 response by inducing a less protective Th17 response. In mouse models, candidate vaccines which provoke a Th1 response can accelerate the clearance of gonococcus from the mouse female genital tract. Human studies indicate that natural infection often induces a limited immune response, with modest antibody responses, which may correlate with the clinical severity of gonococcal disease. Studies of cytokine responses to gonococcal infection in humans provide conflicting evidence as to whether infection induces an IL-17 response. However, there is evidence for limited induction of protective immunity from a study of female sex workers in Kenya. A controlled human infection model (CHIM) has been used to examine the immune response to gonococcal infection in male volunteers, but has not to date demonstrated protection against re-infection. Correlates of protection for gonorrhea are lacking, which has hampered the progress towards developing a successful vaccine. However, the finding that the Neisseria meningitidis serogroup B vaccines, elicit cross-protection against gonorrhea has invigorated the gonococcal vaccine field. More studies of infection in humans, either natural infection or CHIM studies, are needed to understand better gonococcal protective immunity.

Cross protection to SARS-CoV-2 variants in hamsters with naturally-acquired immunity.

Since SARS-CoV-2 was first reported in late 2019, multiple variations of the original virus have emerged. Each variant harbors accumulations of mutations, particularly within the spike glycoprotein, that are associated with increased viral transmissibility and escape immunity. The different mutations in the spike protein of different variants shape the subsequent antibody and T cell responses, such that exposure to different spike proteins can result in reduced or enhanced responses to heterologous variants further down the line. Globally, people have been exposed and re-exposed to multiple variations of the Ancestral strain, including the five variants of concerns. Studies have shown that the protective immune response of an individual is influenced by which strain or combination of strains they are exposed to. The initial exposure to a specific strain may also shape their subsequent immune patterns and response to later infections with a heterologous virus. Most immunological observations were carried out early during the pandemic when the Ancestral strain was circulating. However, SARS-CoV-2 variants exhibit varying patterns of disease severity, waning immunity, immune evasion and sensitivity to therapeutics. Here we investigated the cross-protection in hamsters previously infected with a variant of concern (VOC) and subsequently re-infected with a heterologous variant. We also determined if cross-protection and immunity were dependent on the specific virus to which the hamster was first exposed. We further profiled the host cytokine response induced by each SARS-CoV-2 variants as well as subsequent to re-infection. A comparative analysis of the three VOCs revealed that Alpha variant was the most pathogenic VOC to emerge. We showed that naturally acquired immunity protected hamsters from subsequent re-infection with heterologous SARS-CoV-2 variant, regardless which variant the animal was first exposed to. Our study supports observations that heterologous infection of different SARS-CoV-2 variants do not exacerbate disease in subsequent re-infections. The continual emergence of new SARS-CoV-2 variants mandates a better understanding of cross-protection and immune imprinting in infected individuals. Such information is essential to guide vaccine strategy and public policy to emerging SARS-CoV-2 VOCs and future novel pandemic coronaviruses.

Comparison of Immune Responses between Inactivated and mRNA SARS-CoV-2 Vaccines Used for a Booster Dose in Mice.

A large amount of real-world data suggests that the emergence of variants of concern (VOCs) has brought new challenges to the fight against SARS-CoV-2 because the immune protection elicited by the existing coronavirus disease 2019 (COVID-19) vaccines was weakened. In response to the VOCs, it is necessary to advocate for the administration of booster vaccine doses to extend the effectiveness of vaccines and enhance neutralization titers. In this study, the immune effects of mRNA vaccines based on the WT (prototypic strain) and omicron (B1.1.529) strains for use as booster vaccines were investigated in mice. It was determined that with two-dose inactivated vaccine priming, boosting with mRNA vaccines could elevate IgG titers, enhance cell-mediated immunity, and provide immune protection against the corresponding variants, but cross-protection against distinct strains was inferior. This study comprehensively describes the differences in the mice boosted with mRNA vaccines based on the WT strain and the omicron strain, a harmful VOC that has resulted in a sharp rise in the number of infections, and reveals the most efficacious vaccination strategy against omicron and future SARS-CoV-2 variants.

Comparison of the cross-protection of PPRSV sublineage 8.7 MLV vaccines against the recombinant NADC30-like strain.

The emergence of recombinant porcine reproductive and respiratory syndrome virus (PRRSV) has caused a substantial threat to the swine industry in recent years. However, the protective efficacy of different sublineage 8.7 PRRSV modified-live virus (MLV) vaccines against emerging strains were still obscure. In this study, a broad epidemiological investigation of PRRSV showed the prevalence of NADC30-like strain increased in Shandong Province, China from 2018 to 2020. Through piglet trial for vaccination and challenge with recombinant NADC30-like SDlz1601 strain, CH-1R MLV vaccine showed better protective effect than JXA1-R and TJM-F92 MLV vaccines in terms of clinical score and pathological observation. Moreover, all three MLV vaccines could reduce virus loads in the serum of piglets. This study provides valuable insights into the prevalence of the NADC30-like strain and the protective effect of PRRS MLV vaccines against recombinant NADC30-like strains, which could help to improve the prevention and control of PRRSV infections.

Possible Mpox Protection from Smallpox Vaccine-Generated Antibodies among Older Adults.

Smallpox vaccination may confer cross-protection to mpox. We evaluated vaccinia virus antibodies in 162 persons ≥50 years of age in Spain; 68.5% had detectable antibodies. Highest coverage (78%) was among persons 71-80 years of age. Low antibody levels in 31.5% of this population indicates that addressing their vaccination should be a priority.

Generation of Attenuated Mutants of East Asian Passiflora Virus for Disease Management by Cross Protection.

East Asian passiflora virus (EAPV) seriously affects passionfruit production in Taiwan and Vietnam. In this study, an infectious clone of the EAPV Taiwan strain (EAPV-TW) was constructed, and EAPV-TWnss, with an nss tag attached to its helper component-protease (HC-Pro), was generated for monitoring the virus. Four conserved motifs of EAPV-TW HC-Pro were manipulated to create single mutations of F8I (simplified as I8), R181I (I181), F206L (L206), and E397N (N397) and double mutations of I8I181, I8L206, I8N397, I181L206, I181N397, and L206N397. Four mutants, EAPV I8I181, I8N397, I181L206, and I181N397, infected Nicotiana benthamiana and yellow passionfruit plants without conspicuous symptoms. Mutants EAPV I181N397 and I8N397 were stable after six passages in yellow passionfruit plants and expressed a zigzag pattern of accumulation dynamic, typical of beneficial protective viruses. An agroinfiltration assay indicated that the RNA silencing suppression capabilities of the four double mutated HC-Pros are significantly reduced. Mutant EAPV I181N397 accumulated the highest level of the small interfering RNA at 10 days postinoculation (dpi) in N. benthamiana plants, then dropped to background levels after 15 dpi. In both N. benthamiana and yellow passionfruit plants, EAPV I181N397 conferred complete cross protection (100%) against severe EAPV-TWnss, as defined by no severe symptoms and absence of the challenge virus, checked by Western blotting and reverse transcription PCR. Mutant EAPV I8N397 provided high degrees of complete protection against EAPV-TWnss in yellow passionfruit plants (90%) but not in N. benthamiana plants (0%). Both mutants showed complete protection (100%) against the Vietnam severe strain EAPV-GL1 in passionfruit plants. Thus, the mutants EAPV I181N397 and I8N397 have excellent potential for controlling EAPV in Taiwan and Vietnam. [Formula: see text] Copyright © 2023 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Immunity to Seasonal Coronavirus Spike Proteins Does Not Protect from SARS-CoV-2 Challenge in a Mouse Model but Has No Detrimental Effect on Protection Mediated by COVID-19 mRNA Vaccination.

Seasonal coronaviruses have been circulating widely in the human population for many years. With increasing age, humans are more likely to have been exposed to these viruses and to have developed immunity against them. It has been hypothesized that this immunity to seasonal coronaviruses may provide partial protection against infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and it has also been shown that coronavirus disease 2019 (COVID-19) vaccination induces a back-boosting effects against the spike proteins of seasonal betacoronaviruses. In this study, we tested if immunity to the seasonal coronavirus spikes from OC43, HKU1, 229E, or NL63 would confer protection against SARS-CoV-2 challenge in a mouse model, and whether pre-existing immunity against these spikes would weaken the protection afforded by mRNA COVID-19 vaccination. We found that mice vaccinated with the seasonal coronavirus spike proteins had no increased protection compared to the negative controls. While a negligible back-boosting effect against betacoronavirus spike proteins was observed after SARS-CoV-2 infection, there was no negative original antigenic sin-like effect on the immune response and protection induced by SARS-CoV-2 mRNA vaccination in animals with pre-existing immunity to seasonal coronavirus spike proteins. IMPORTANCE The impact that immunity against seasonal coronaviruses has on both susceptibility to SARS-CoV-2 infection as well as on COVID-19 vaccination is unclear. This study provides insights into both questions in a mouse model of SARS-CoV-2.

Evaluation of cross-protection between S. Pneumoniae serotypes 35B and 29 in a mouse model.

Pneumococcal conjugate vaccines (PCVs) have reduced vaccine-type pneumococcal disease but in turn have also resulted in replacement with non-vaccine serotypes. One such serotype, 35B, a multidrug resistant type, has been associated with an increase in disease. Mice were immunized intramuscularly with monovalent pneumococcal polysaccharide 35B conjugated to CRM197 containing aluminum phosphate adjuvant on days 0, 14, and 28. Pneumococcal enzyme-linked immunosorbent assay, opsonophagocytic killing assays, and competition OPA were performed for STs 35B and 29 to measure serotype-specific binding and functional antibodies. On day 52, mice were intratracheally challenged with S. pneumoniae ST29 to evaluate cross-protection. 35B-CRM197 immunized mice had binding and functional antibodies to both PnPs 35B and 29. 35B-CRM197 immunized mice were 100% protected from IT challenge with S. pneumoniae ST29 as compared to 30% survival in the naïve group. Future vaccines containing polysaccharide 35B, such as the investigational 21-valent PCV, V116, may provide cross protection against the non-vaccine serotype 29 due to structural similarity.

Cross-protective antibodies against common endemic respiratory viruses.

Respiratory syncytial virus (RSV), human metapneumovirus (HMPV), and human parainfluenza virus types one (HPIV1) and three (HPIV3) can cause severe disease and death in immunocompromised patients, the elderly, and those with underlying lung disease. A protective monoclonal antibody exists for RSV, but clinical use is limited to high-risk infant populations. Hence, therapeutic options for these viruses in vulnerable patient populations are currently limited. Here, we present the discovery, in vitro characterization, and in vivo efficacy testing of two cross-neutralizing monoclonal antibodies, one targeting both HPIV3 and HPIV1 and the other targeting both RSV and HMPV. The 3 × 1 antibody is capable of targeting multiple parainfluenza viruses; the MxR antibody shares features with other previously reported monoclonal antibodies that are capable of neutralizing both RSV and HMPV. We obtained structures using cryo-electron microscopy of these antibodies in complex with their antigens at 3.62 Å resolution for 3 × 1 bound to HPIV3 and at 2.24 Å for MxR bound to RSV, providing a structural basis for in vitro binding and neutralization. Together, a cocktail of 3 × 1 and MxR could have clinical utility in providing broad protection against four of the respiratory viruses that cause significant morbidity and mortality in at-risk individuals.

Anti-neuraminidase and anti-hemagglutinin immune serum can confer inter-lineage cross protection against recent influenza B.

Influenza B viruses (IBV) are responsible for a considerable part of the burden caused by influenza virus infections. Since their emergence in the 1980s, the Yamagata and Victoria antigenic lineages of influenza B circulate in alternate patterns across the globe. Furthermore, their evolutionary divergence and the appearance of new IBV subclades complicates the prediction of future influenza vaccines compositions. It has been proposed that the addition of the neuraminidase (NA) antigen could potentially induce a broader protection and compensate for hemagglutinin (HA) mismatches in the current vaccines. Here we show that anti-NA and -HA sera against both Victoria and Yamagata lineages have limited inter-lineage cross-reactivity. When transferred to mice prior to infection with a panel of IBVs, anti-NA sera were as potent as anti-HA sera in conferring protection against homologous challenge and, in some cases, conferred superior protection against challenge with heterologous IBV strains.

Mouse models susceptible to HCoV-229E and HCoV-NL63 and cross protection from challenge with SARS-CoV-2.

Human coronavirus 229E (HCoV-229E) and NL63 (HCoV-NL63) are endemic causes of upper respiratory infections such as the "common cold" but may occasionally cause severe lower respiratory tract disease in the elderly and immunocompromised patients. There are no approved antiviral drugs or vaccines for these common cold coronaviruses (CCCoV). The recent emergence of COVID-19 and the possible cross-reactive antibody and T cell responses between these CCCoV and SARS-CoV-2 emphasize the need to develop experimental animal models for CCCoV. Mice are an ideal experimental animal model for such studies, but are resistant to HCoV-229E and HCoV-NL63 infections. Here, we generated 229E and NL63 mouse models by exogenous delivery of their receptors, human hAPN and hACE2 using replication-deficient adenoviruses (Ad5-hAPN and Ad5-hACE2), respectively. Ad5-hAPN- and Ad5-hACE2-sensitized IFNAR-/- and STAT1-/- mice developed pneumonia characterized by inflammatory cell infiltration with virus clearance occurring 7 d post infection. Ad5-hAPN- and Ad5-hACE2-sensitized mice generated virus-specific T cells and neutralizing antibodies after 229E or NL63 infection, respectively. Remdesivir and a vaccine candidate targeting spike protein of 229E and NL63 accelerated viral clearance of virus in these mice. 229E- and NL63-infected mice were partially protected from SARS-CoV-2 infection, likely mediated by cross-reactive T cell responses. Ad5-hAPN- and Ad5-hACE2-transduced mice are useful for studying pathogenesis and immune responses induced by HCoV-229E and HCoV-NL63 infections and for validation of broadly protective vaccines, antibodies, and therapeutics against human respiratory coronaviruses including SARS-CoV-2.