Viral tegument proteins restrict cGAS-DNA phase separation to mediate immune evasion.

DNA-induced liquid-liquid phase separation of cyclic GMP-AMP synthase (cGAS) triggers a potent response to detect pathogen infection and promote innate immune signaling. Whether and how pathogens manipulate cGAS-DNA condensation to mediate immune evasion is unknown. We report the identification of a structurally related viral tegument protein family, represented by ORF52 and VP22 from gamma- and alpha-herpesvirinae, respectively, that employs a conserved mechanism to restrict cGAS-DNA phase separation. ORF52/VP22 proteins accumulate into, and effectively disrupt, the pre-formed cGAS-DNA condensation both in vitro and in cells. The inhibition process is dependent on DNA-induced liquid-liquid phase separation of the viral protein rather than a direct interaction with cGAS. Moreover, highly abundant ORF52 proteins carried within viral particles are able to target cGAS-DNA phase separation in early infection stage. Our results define ORF52/VP22-type tegument proteins as a family of inhibitors targeting cGAS-DNA phase separation and demonstrate a mechanism for how viruses overcome innate immunity.

Highly Sensitive and Specific Multiplex Antibody Assays To Quantify Immunoglobulins M, A, and G against SARS-CoV-2 Antigens.

Reliable serological tests are required to determine the prevalence of antibodies against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and to characterize immunity to the disease in order to address key knowledge gaps in the coronavirus disease 2019 (COVID-19) pandemic. Quantitative suspension array technology (qSAT) assays based on the xMAP Luminex platform overcome the limitations of rapid diagnostic tests and enzyme-linked immunosorbent assays (ELISAs) with their higher precision, dynamic range, throughput, miniaturization, cost-efficiency, and multiplexing capacity. We developed three qSAT assays for IgM, IgA, and IgG against a panel of eight SARS-CoV-2 antigens, including spike protein (S), nucleocapsid protein (N), and membrane protein (M) constructs. The assays were optimized to minimize the processing time and maximize the signal-to-noise ratio. We evaluated their performances using 128 prepandemic plasma samples (negative controls) and 104 plasma samples from individuals with SARS-CoV-2 diagnosis (positive controls), of whom 5 were asymptomatic, 51 had mild symptoms, and 48 were hospitalized. Preexisting IgG antibodies recognizing N, M, and S proteins were detected in negative controls, which is suggestive of cross-reactivity to common-cold coronaviruses. The best-performing antibody/antigen signatures had specificities of 100% and sensitivities of 95.78% at ≥14 days and 95.65% at ≥21 days since the onset of symptoms, with areas under the curve (AUCs) of 0.977 and 0.999, respectively. Combining multiple markers as assessed by qSAT assays has the highest efficiency, breadth, and versatility to accurately detect low-level antibody responses for obtaining reliable data on the prevalence of exposure to novel pathogens in a population. Our assays will allow gaining insights into antibody correlates of immunity and their kinetics, required for vaccine development to combat the COVID-19 pandemic.

Generation and immunogenicity analysis of recombinant classical swine fever virus glycoprotein E2 and Erns expressed in baculovirus expression system.

Classical swine fever (CSF) caused by the classical swine fever virus (CSFV) is a highly contagious swine disease resulting in large economical losses worldwide. The viral envelope glycoprotein E2 and Erns are major targets for eliciting antibodies against CSFV in infected animals. In this report, the glycoprotein E2 and Erns were expressed using the baculovirus system and their protective immunity in rabbits were tested. Twenty CSFV seronegative rabbits were randomly divided into five groups. Each rabbit was intramuscularly immunized with CSFV-E2, CSFV-Erns, or their combination (CSFV-E2 + Erns). Besides, a commercial CSFV vaccine (C-strain) and PBS were used as positive or negative controls, respectively. Four weeks after the second immunization, all the rabbits were challenged with 100 RID50 of CSFV C-strain. High levels of CSFV E2-specific antibody, neutralizing antibody and cellular immune responses to CSFV were elicited in the rabbits inoculated with C-strain, CSFV-E2, and CSFV-E2 + Erns. And the rabbits inoculated with the three vaccines received complete protection against CSFV C-strain. However, no neutralizing antibody was detected in the Erns vaccinated rabbits and the rabbits exhibited fever typical of CSFV, suggesting the Erns alone is not able to induce a protective immune response. Taken together, while the Erns could not confer protection against CSFV, E2 and E2 + Erns could not only elicit humoral and cell-mediated immune responses but also confer complete protection against CSFV C-strain in rabbits.

Co-expression of self-cleaved multiple proteins derived from Porcine Reproductive and Respiratory Syndrome Virus by bi-cistronic and tri-cistronic DNA vaccines.

Porcine Reproductive and Respiratory Syndrome caused by Porcine Reproductive and Respiratory Syndrome Virus (PRRSV) remains one of the important diseases in swine industry. A vaccine that is safe, effective and also elicit broad immune response against multiple antigens is desirable. In this study, we developed multi-cistronic DNA vaccines capable of co-expressing multiple structural proteins derived from PRRSV. To preserve the structure and function of each antigen protein, we employed self-cleaving 2A peptides to mediate separation of multiple proteins expressed by multi-cistronic genes. Six bi-cistronic genes encoding PRRSV GP5 and M proteins were generated, by which each construct contains different 2A sequences derived from Foot-and-mouth disease virus (F2A), porcine teschovirus-1 (P2A) and Thosea asigna virus (T2A) either with or without furin cleavage site (Fu). Vectored by the mammalian expression plasmid pTH, all six bi-cistronic genes co-expressed the proteins GP5 and M at comparable level. Importantly, all six types of 2A sequences could mediate a complete self-cleavage of the GP5 and M. We next generated tri-cistronic DNA vaccines co-expressing the PRRSV proteins GP5, M and N. All homologous and heterologous combinations of P2A and F2A in tri-cistronic genes yielded a complete self-cleavage of the GP5, M and N proteins. Our study reports a success in co-expression of multiple PRRSV structural proteins in discrete form from a single vaccine and confirms feasibility of developing one single vaccine that provides broad immune responses against PRRSV.

Inactivated infectious pancreatic necrosis virus (IPNV) vaccine and E.coli-expressed recombinant IPNV-VP2 subunit vaccine afford protection against IPNV challenge in rainbow trout.

Infectious pancreatic necrosis (IPN) is a highly contagious disease causing high mortality in juvenile trouts. Since there is no effective way to treatment against IPNV, early diagnosis and prevention play an important role in combating the disease. The different types of IPNV vaccines (inactive, live, recombinant, DNA, etc) have been produced from local isolates and have been used in developed countries. In Turkey, there is no commercial licensed vaccines against IPNV. Due to this reason, IPNV vaccine is needed in Turkey. The production of recombinant VP2 subunit vaccine (IPNV-VP2) and inactivated whole particle virus vaccine (IPNV-WPV) were attempted from selected isolate belong to sp serotype. For this purpose; the virus was produced in RTG-2 cell line and RT-PCR amplification was performed by using primers with restriction enzymes. The whole VP2 gene was cloned into a plasmid vector and VP2 was expressed by using E. coli expression system. A trial was conducted to determine the immunity ability of IPNV-VP2 and IPNV-WPV in rainbow trout. According to the SN50 assay, the IPNV-WPV stimulates immune response faster than the IPNV-VP2 vaccine. Besides, the relative percent of Survive (RPS) was detected as 79% in fish vaccinated with IPNV-WPV and 70% in fish vaccinated with IPNV-VP2. Thus, we can say that the recombinant vaccine of IPNV-VP2 is almost protected against IPNV infection as well as the inactive vaccine.

The rescue and selection of thermally stable type O vaccine candidate strains of foot-and-mouth disease virus.

Inactivated foot-and-mouth disease virus (FMDV) vaccines have been used widely to control foot-and-mouth disease (FMD). However, the virions (146S) of this virus are easily dissociated into pentamer subunits (12S), which limits the immune protective efficacy of inactivated vaccines when the temperature is higher than 30 °C. A cold-chain system can maintain the quality of the vaccines, but such systems are usually not reliable in limited-resource settings. Thus, it is imperative to improve the thermostability of vaccine strains to guarantee the quality of the vaccines. In this study, four recombinant FMDV strains containing single or multiple amino acid substitutions in the structural proteins were rescued using a previously constructed FMDV type O full-length infectious clone (pO/DY-VP1). We found that single or multiple amino acid substitutions in the structural proteins affected viral replication to different degrees. Furthermore, the heat and acid stability of the recombinant viruses was significantly increased when compared with the parental virus. Three thermally stable recombinant viruses (rHN/DY-VP1Y2098F, rHN/DY-VP1V2090A-S2093H, and rHN/DY-VP1V2090A-S2093H-Y2098F) were prepared as inactivated vaccines to immunize pigs. Blood samples were collected every week to prepare sera, and a virus neutralization test showed that the substitutions S2093H and Y2098F, separately or in combination, did not affect the immunogenicity of the virus, but the Y2098F mutation increased the thermostability significantly (p < 0.05). Therefore, the rHN/DY-VP1Y2098F mutant should be considered for use in future vaccines.

Assessing the antigenicity of different VP3 regions of infectious bursal disease virus in chickens from South Brazil.

BACKGROUND: Infectious bursal disease (IBD), also known as Gumboro disease, is a viral infection that causes mortality and immunosuppression in chickens (Gallus gallus). VP2 and VP3 are the major structural viral capsid components and are the most immunogenic proteins of IBD virus (IBDV). Reliable diagnostic tests using VP2 and VP3 produced in heterologous systems are important tools to control this infection. One advantage of an IBD diagnostic based on VP3, over those that use VP2, is that VP3 has linear epitopes, enabling its production in bacteria. RESULTS: We tested the suitability of recombinant VP3 (rVP3) as a diagnostic reagent in an enzyme-linked immunosorbent assay (ELISA). Compared with a commercial test, rVP3 ELISA showed high sensitivity and specificity as a diagnostic tool for vaccinated animals. In addition, rVP3, but not the commercial ELISA, was able to detect antibodies in nonvaccinated chickens, probably developed against circulating IBDV strains. It was possible the assessment of VP3 regions antigenicity using chicken antisera. CONCLUSIONS: The full-length recombinant VP3 can be used to assess post vaccination immunological status of chickens and its production is feasible and inexpensive. The evaluation of VP3 regions as candidates for general use in the diagnosis of IBD in chickens should be conducted with caution. Our work was the first to identify several regions of VP3 recognized by chicken antibodies.

Detection of Antibodies against Hepatitis A Virus (HAV) by a Surface Plasmon Resonance (SPR) Biosensor: A New Diagnosis Tool Based on the Major HAV Capsid Protein VP1 (SPR-HAVP1).

Hepatitis A (HA) is an acute human infectious disease caused by a positive single-stranded RNA virus (HAV). It is mainly acquired through the fecal-oral route and is primarily spread by contact between people and exposure to contaminated water and food. Recently, large outbreaks of HA have been reported by low and moderate endemicity countries, emphasizing its importance in public health and the need for rapid and large-scale diagnostic tests to support public health decisions on HA. This work proposes a new tool for HAV diagnosis based on the association of surface plasmonic resonance with major capsid protein VP1 (SPR-HAVP1 assay), detecting IgM antibodies for HAV in human serum samples. Structural analyses of VP1 B-lymphocyte epitopes showed continuous and discontinuous epitopes. The discontinuous epitopes were identified in the N-terminal region of the VP1 protein. Both epitope types in the VP1 protein were shown by the reactivity of VP1 in native and denaturing conditions to IgM anti-HAV, which was favorable to tests of VP1 in the SPR assays. SPR-HAVP1 assays showed good performance in the detection of IgM polyclonal antibody anti-HAV. These assays were performed using a COOH5 sensor chip functionalized with VP1 protein. The sensorgram record showed a significant difference between positive and negative serum samples, which was confirmed by analysis of variation of initial and final dissociation values through time (ΔRUd/t). The data gathered here are unequivocal evidence that the SPR-HAVP1 strategy can be applied to detect IgM antibodies in human serum positive to the HAV. This is a new tool to be explored to diagnose human HAV infections.

Population-Predicted MHC Class II Epitope Presentation of SARS-CoV-2 Structural Proteins Correlates to the Case Fatality Rates of COVID-19 in Different Countries.

We observed substantial differences in predicted Major Histocompatibility Complex II (MHCII) epitope presentation of SARS-CoV-2 proteins for different populations but only minor differences in predicted MHCI epitope presentation. A comparison of this predicted epitope MHC-coverage revealed for the early phase of infection spread (till day 15 after reaching 128 observed infection cases) highly significant negative correlations with the case fatality rate. Specifically, this was observed in different populations for MHC class II presentation of the viral spike protein (p-value: 0.0733 for linear regression), the envelope protein (p-value: 0.023), and the membrane protein (p-value: 0.00053), indicating that the high case fatality rates of COVID-19 observed in some countries seem to be related with poor MHC class II presentation and hence weak adaptive immune response against these viral envelope proteins. Our results highlight the general importance of the SARS-CoV-2 structural proteins in immunological control in early infection spread looking at a global census in various countries and taking case fatality rate into account. Other factors such as health system and control measures become more important after the early spread. Our study should encourage further studies on MHCII alleles as potential risk factors in COVID-19 including assessment of local populations and specific allele distributions.

Novel monoclonal antibodies against Australian strains of negeviruses and insights into virus structure, replication and host -restriction.

We report the isolation of Australian strains of Bustos virus and Ngewotan virus, two insect-specific viruses in the newly identified taxon Negevirus, originally isolated from Southeast Asian mosquitoes. Consistent with the expected insect-specific tropism of negeviruses, these isolates of Ngewotan and Bustos viruses, alongside the Australian negevirus Castlerea virus, replicated exclusively in mosquito cells but not in vertebrate cells, even when their temperature was reduced to 34 °C. Our data confirmed the existence of two structural proteins, putatively one membrane protein forming the majority of the virus particle, and one glycoprotein forming a projection on the apex of the virions. We generated and characterized 71 monoclonal antibodies to both structural proteins of the two viruses, most of which were neutralizing. Overall, these data increase our knowledge of negevirus mechanisms of infection and replication in vitro.

Oral immunization with recombinant Lactobacillus casei displayed AHA1-CK6 and VP2 induces protection against infectious pancreatic necrosis in rainbow trout (Oncorhynchus mykiss).

Infectious pancreatic necrosis virus (IPNV) primarily infects larvae and young salmonid with serious economic losses, which causes haemorrhage and putrescence of hepatopancreas. To develop a more effective oral vaccine against IPNV infection, the aeromonas hydrophila adhesion (AHA1) gene was used as a targeting molecule for intestinal epithelial cells. A genetically engineered Lactobacillus casei (pPG-612-AHA1-CK6-VP2/L. casei 393) was constructed to express the AHA1-CK6-VP2 fusion protein. The expression of interest protein was confirmed by western blotting and the immunogenicity of pPG-612-AHA1-CK6-VP2/L. casei 393 was evaluated. And the results showed that more pPG-612-AHA1-CK6-VP2/L. casei 393 were found in the intestinal mucosal surface of the immunized group. The Lactobacillus-derived AHA1-CK6-VP2 fusion protein could induce the production of serum IgM and skin mucus IgT specific for IPNV with neutralizing activity in rainbow trouts. The levels of IL-1ß, IL-8 and TNF-α isolated from the lymphocytes stimulated by AHA1-CK6-EGFP produced were significantly higher than EGFP group. For transcription levels of IL-1ß, IL-8, CK6, MHC-II, Mx and TNF-1α in the spleen, the result indicated that the adhesion and target chemokine recruit more immune cells to induce cellular immunity. The level of IPNV in the immunized group of pPG-612-AHA1-CK6-VP2/L. casei 393 was significantly lower than that in the control groups. These data indicated that the adhesion and target chemokine could enhance antigen delivery efficiency, which provides a valuable strategy for the development of IPNV recombination Lactobacillus casei oral vaccine in the future.

Generation of recombinant VP3 protein of infectious bursal disease virus in three different expression systems, antigenic analysis of the obtained polypeptides and development of an ELISA test.

Infectious bursal disease virus (IBDV), which infects young chickens, is one of the most important pathogens that harm the poultry industry. Evaluation of the immune status of birds before and after vaccination is of great importance for controlling the disease caused by this virus. Therefore, the development of low-cost and easy-to-manufacture test systems for IBDV antibody detection remains an urgent issue. In this study, three expression systems (bacteria, yeast, and human cells) were used to produce recombinant VP3 protein of IBDV. VP3 is a group-specific antigen and hence may be a good candidate for use in diagnostic tests. Comparison of the antigenic properties of the obtained polypeptides showed that the titres of antibodies raised in chickens against bacteria- or human-cell-derived recombinant VP3 were high, whereas the antibody level against yeast-derived recombinant VP3 was low. The results of an enzyme-linked immunosorbent assay (ELISA) of sera from IBDV-infected chickens demonstrated that the recombinant VP3 produced in E. coli would be the best choice for use in test systems.

B Cell-Intrinsic MyD88 Signaling Promotes Initial Cell Proliferation and Differentiation To Enhance the Germinal Center Response to a Virus-like Particle.

Although TLR signaling in B cells has been implicated in the germinal center (GC) responses during viral infections and autoimmune diseases, the underlying mechanism is unclear. Bacterial phage Qß-derived virus-like particle (Qß-VLP) contains TLR ligands, which can enhance Qß-VLP-induced Ab response, including GC response, through TLR/MyD88 signaling in B cells. In this study, by examining Ag-specific B cell response to Qß-VLP, we found that lack of B cell MyD88 from the beginning of the immune response led to a more severe defect in the GC scale than abolishing MyD88 at later time points of the immune response. Consistently, B cell-intrinsic MyD88 signaling significantly enhanced the initial proliferation of Ag-specific B cells, which was accompanied with a dramatic increase of plasma cell generation and induction of Bcl-6+ GC B cell precursors. In addition, B cell-intrinsic MyD88 signaling promoted strong T-bet expression independent of IFN-γ and led to the preferential isotype switching to IgG2a/c. Thus, by promoting the initial Ag-specific B cell proliferation and differentiation, B cell-intrinsic MyD88 signaling enhanced both T-independent and T-dependent Ab responses elicited by Qß-VLP. This finding will provide additional insight into the role of TLR signaling in antiviral immunity, autoimmune diseases, and vaccine design.

Development of a recombinant VP2 vaccine for the prevention of novel variant strains of infectious bursal disease virus.

Since 2017, novel variant strains of infectious bursal disease virus (nvIBDV) have been detected in China, while the current vaccines on the market against very virulent IBDV have limited protection against this subtype virus. In this context, a strain of the virus has been isolated, and sequencing alignment and bird regression experiments showed that the virus was IBDV, belonging to the nvIBDV subtype (and named IBDV FJ-1812). Furthermore, the Escherichia coli expression system was used to successfully express soluble nvIBDV rVP2, which is specifically recognized by an anti-IBDV standard serum and anti-nvIBDV positive serum, and could be assembled into 14 - 17 nm virus-like particles. Based on the purified nvIBDV rVP2, we developed an IBDV FJ-1812 VP2 VLP vaccine at a laboratory scale to evaluate protection by this vaccine; in addition, we also prepared an IBDV JZ 3/02 VP2 subunit vaccine targeting very virulent IBDV and evaluated its cross-protection against nvIBDV. Results of bird experiments showed that the nvIBDV rVP2 vaccine could induce high titres of specific antibodies, completely protect the bursa of Fabricius from viral infection, and provide 100% immune protection to SPF and Ross 308 broiler chickens. Furthermore, the IBDV JZ 3/02 VP2 subunit vaccine targeting very virulent IBDV could provide 60% protection for SPF chickens and 80% protection for Ross 308 broiler chickens. This report provides important technical supports for the prevention and control of nvIBDV in the future.

Characterization of the highly immunogenic VP2 protrusion domain as a diagnostic antigen for members of Birnaviridae family.

Birnaviridae is a family of viruses (birnaviruses) which consists of four genera, members of which cause diseases in fish, birds, mollusks, and insects. The genome of birnaviruses encodes the highly immunogenic VP2 capsid protein. In order to demonstrate that the VP2 protein can be exploited as a diagnostic antigen for birnaviruses, we developed a lateral flow assay based on the surface-exposed VP2 protrusion domain of a representative birnavirus, infectious bursal disease virus (IBDV) of serotype 1 which causes the highly devastating infectious bursal disease in chickens. The biophysical characterization of the purified domain reveals that the domain predominantly consists of ß-sheets, exists in a trimeric form, and remains folded at high temperatures, making it suitable for diagnostic purposes. Owing to its highly immunogenic nature and excellent biophysical properties, we employed the VP2 protrusion domain in a gold nanoparticle-based lateral flow assay for rapid detection of anti-IBDV antibodies in serum samples of infected chickens. Our results indicate that the domain binds anti-IBDV antibodies with high specificity during laboratory testing and on-site testing. The lateral flow assay reported here yields comparable results in a qualitative manner as obtained through a commercial enzyme-linked immunosorbent assay (ELISA). As VP2 is a common capsid protein of birnaviruses, the lateral flow assay can be generalized for other birnaviruses, and members of Tetraviridae and Nodaviridae families which contain homologous VP2 capsid proteins.

Proteomics Analysis Reveals That Structural Proteins of the Virion Core and Involved in Gene Expression Are the Main Source for HLA Class II Ligands in Vaccinia Virus-Infected Cells.

Protective cellular and humoral immune responses require previous recognition of viral antigenic peptides complexed with human leukocyte antigen (HLA) class II molecules on the surface of the antigen presenting cells. The HLA class II-restricted immune response is important for the control and the clearance of poxvirus infection including vaccinia virus (VACV), the vaccine used in the worldwide eradication of smallpox. In this study, a mass spectrometry analysis was used to identify VACV ligands bound to HLA-DR and -DP class II molecules present on the surface of VACV-infected cells. Twenty-six naturally processed viral ligands among the tens of thousands of cell peptides bound to HLA class II proteins were identified. These viral ligands arose from 19 parental VACV proteins: A4, A5, A18, A35, A38, B5, B13, D1, D5, D7, D12, D13, E3, E8, H5, I2, I3, J2, and K2. The majority of these VACV proteins yielded one HLA ligand and were generated mainly, but not exclusively, by the classical HLA class II antigen processing pathway. Medium-sized and abundant proteins from the virion core and/or involved in the viral gene expression were the major source of VACV ligands bound to HLA-DR and -DP class II molecules. These findings will help to understand the effectiveness of current poxvirus-based vaccines and will be important in the design of new ones.

Reduced Cell-Associated DNA and Improved Viral Control in Macaques following Passive Transfer of a Single Anti-V2 Monoclonal Antibody and Repeated Simian/Human Immunodeficiency Virus Challenges.

A high level of V1V2-specific IgG antibodies (Abs) in vaccinees' sera was the only independent variable that correlated with a reduced risk of human immunodeficiency virus (HIV) acquisition in the RV144 clinical trial. In contrast, IgG avidity, antibody neutralization, and antibody-dependent cellular cytotoxicity each failed as independent correlates of infection. Extended analyses of RV144 samples demonstrated the antiviral activities of V1V2-specific vaccine-induced antibodies. V2-specific antibodies have also been associated with protection from simian immunodeficiency virus (SIV), and the V2i-specific subset of human monoclonal antibodies (MAbs), while poor neutralizers, mediates Fc-dependent antiviral functions in vitro The objective of this study was to determine the protective efficacy of a V2i-specific human MAb, 830A, against mucosal simian/human immunodeficiency virus (SHIV) challenge. V2i MAb binding sites overlap the integrin binding site in the V2 region and are similar to the epitopes bound by antibodies associated with reduced HIV infection rates in RV144. Because the IgG3 subclass was a correlate of reduced infection rates in RV144, we compared passive protection by both IgG1 and IgG3 subclasses of V2i MAb 830A. This experiment represents the first in vivo test of the hypothesis emanating from RV144 and SIV studies that V2i Abs can reduce the risk of infection. The results show that passive transfer with a single V2i MAb, IgG1 830A, reduced plasma and peripheral blood mononuclear cell (PBMC) virus levels and decreased viral DNA in lymphoid tissues compared to controls, but too few animals remained uninfected to achieve significance in reducing the risk of infection. Based on these findings, we conclude that V2i antibodies can impede virus seeding following mucosal challenge, resulting in improved virus control.IMPORTANCE Since the results of the HIV RV144 clinical trial were reported, there has been significant interest in understanding how protection was mediated. Antibodies directed to a subregion of the envelope protein called V1V2 were directly correlated with a reduced risk, and surprisingly low virus neutralization was observed. To determine whether these antibodies alone could mediate protection, we used a human monoclonal antibody directed to V2 with properties similar to those elicited in the vaccine trial for passive infusions in rhesus macaques and challenge with SHIV. The single V2 antibody at the dose given did not significantly reduce the number of infections, but there was a significant reduction in the seeding of virus to the lymph nodes and a decrease in plasma viremia in the HIV antibody-infused macaques compared with the control antibody-infused animals. This finding shows that V2 antibodies mediate antiviral activities in vivo that could contribute to a protective HIV vaccine.

Herpes Simplex Virus 1 Tegument Protein VP22 Abrogates cGAS/STING-Mediated Antiviral Innate Immunity.

Cytosolic DNA arising from intracellular pathogens is sensed by cyclic GMP-AMP synthase (cGAS) and triggers a powerful innate immune response. However, herpes simplex virus 1 (HSV-1), a double-stranded DNA virus, has developed multiple mechanisms to attenuate host antiviral machinery and facilitate viral infection and replication. In the present study, we found that HSV-1 tegument protein VP22 acts as an inhibitor of cGAS/stimulator of interferon genes (cGAS/STING)-mediated production of interferon (IFN) and its downstream antiviral genes. Our results showed that ectopic expression of VP22 decreased cGAS/STING-mediated IFN-ß promoter activation and IFN-ß production. Infection with wild-type (WT) HSV-1, but not VP22-deficient virus (ΔVP22), inhibited immunostimulatory DNA (ISD)-induced activation of the IFN signaling pathway. Further study showed that VP22 interacted with cGAS and inhibited the enzymatic activity of cGAS. In addition, stable knockdown of cGAS facilitated the replication of ΔVP22 virus but not the WT. In summary, our findings indicate that HSV-1 VP22 acts as an antagonist of IFN signaling to persistently evade host innate antiviral responses.IMPORTANCE cGAS is very important for host defense against viral infection, and many viruses have evolved ways to target cGAS and successfully evade the attack by the immune system of their susceptible host. This study demonstrated that HSV-1 tegument protein VP22 counteracts the cGAS/STING-mediated DNA-sensing antiviral innate immunity signaling pathway by inhibiting the enzymatic activity of cGAS. The findings in this study will expand our understanding of the interaction between HSV-1 replication and the host DNA-sensing signaling pathway.

In silico analysis of four structural proteins of aphthovirus serotypes revealed significant B and T cell epitopes.

Foot and Mouth disease (FMD) is economically devastating, highly contagious transboundry viral disease of livestock with 100% morbidity, rapid spread and severe production losses in animals. The FMDV has seven different serotypes. There is no vaccine that can protect animals from all serotypes. Hence, it is need of the day to develop a vaccine that protects animals from hetrologous challenge. In this study, we used immunoinformatics approach to find T and B-cell epitopes that will help to construct a universal vaccine for FMDV. For this purpose, first we constructed a consensus sequence for four structural proteins (VP1, VP2, VP3 and VP4) of aphthovirus for seven serotypes (A, O, C, Asia1, SAT1, SAT2 and SAT3). Various computational tools were used to perform multiple sequence alignment to identify the conserved regions, generation of consensus sequence through conserved regions, structures prediction and finally prediction of B and T cell epitopes. We predicted 5 B cell and 18 T cell epitopes. Finally a GPGPG spacer was used to join these epitopes to decrease binding affinity around the core binding regions. Hence, our study identified the epitopes which can be used to develop cross protective vaccines against all the fatal strains of Aphthovirus which can easily protect all the serotypes. Though, successful In vivo and In vitro studies are required to determine the genuine strength of our predicted epitopes against the fatal strains of Aphthovirus.

Neutralizing-antibody-mediated protection of chickens against infectious bursal disease via one-time vaccination with inactivated recombinant Lactococcus lactis expressing a fusion protein constructed from the RCK protein of Salmonella enterica and VP2 of infectious bursal disease virus.

BACKGROUND: Infectious bursal disease (IBD) is an acute contagious immunosuppressive disease which lead to acute bursal injury and immune dysfunction in poultry. It has caused heavy economic losses in the commercial poultry industry for many years in worldwide. Attenuated live vaccine has widely used in poultry showing some promising signs against IBDV infection. But it has defects such as generating enhanced virulence and immunosuppression prohibits. Therefore, the development of mucosal vaccines using the food-grade lactic acid bacterium is necessary. Here, we construct a recombinant Lactococcus co-expressing the major IBDV antigens VP2 and RCK protein of Salmonella enterica to prevent IBD. RESULTS: The recombinant fusion protein VP2-RCK was expressed in a soluble and stable form in the cytoplasm of the recombinant Lactococcus lactis. Animal experiments showed that: (1) the survival rates of the injected immunization inactivated recombinant LAB group and oral immunization live recombinant LAB group were 100% and 80%, respectively; (2) ELISA titers of all serum samples from all experimental groups were negative, but high amounts of specific neutralizing antibodies were detected (1:210 to 1:212); and (3) the bursas of the injected immunization inactivated recombinant LAB group did not suffer damage, as confirmed by clinical observation and bursal histopathological examination. Our results indicate that r-L. lactis-OptiVP2-RCK induces a specific neutralizing-antibody-mediated immune response that confers full protection against very-virulent IBDV (vvIBDV) challenge. CONCLUSION: Lactococcus lactis NZ3900 strain and its matching plasmid pNZ8149 could express the recombinant fusion protein VP2-RCK in a soluble form in the cytoplasm. The protective efficacy of r-L. lactis-OptiVP2-RCK (100%) was better than r-L. lactis-OptiVP2 (0%) which prove RCK protein played its unique role. The neutralizing antibodies titers against infectious bursal disease virus via one-time vaccination with inactivated r-L. lactis-OptiVP2-RCK could reach 1:210 to 1:212, but ELISA titers of all serum samples were negative. For this phenomenon, perhaps because of the change of delivery pathway or the spatial structure of fusion protein. We need further study to test these hypotheses.