Limosilactobacillus reuteri DSM 17938 supplementation and SARS-CoV-2 specific antibody response in healthy adults: a randomized, triple-blinded, placebo-controlled trial.

Studies have shown that probiotics can decrease the symptoms of respiratory tract infections as well as increase antibody responses following certain vaccinations. We examined the effect of probiotic supplementation on anti-SARS-CoV-2 specific antibody responses upon SARS-CoV-2 infection as well as after COVID-19 vaccination. In this randomized, triple-blinded, placebo-controlled intervention study with a parallel design, 159 healthy adults without prior SARS-CoV-2 infection or COVID-19 vaccination and any known risk factors for severe COVID-19 were randomly allocated into two study arms. The active treatment arm consumed a probiotic product containing a minimum of 1 × 108 colony-forming units of Limosilactobacillus reuteri DSM 17938 + 10 µg vitamin D3 twice daily for 6 months. The placebo arm consumed identical tablets containing only 10 µg vitamin D3. Anti-SARS-CoV-2 specific antibodies and virus neutralizing antibody titers were analyzed from blood samples collected at baseline, after 3 months, and after 6 months. Differences in serum antibody titers between the two study arms were tested with independent t-test using log-transformed values. In the intention-to-treat (ITT) analysis, SARS-CoV-2 infected individuals in the active treatment arm (n = 6) tended to have higher serum anti-spike IgG (609 [168-1480] BAU/ml vs 111 [36.1-1210] BAU/ml, p = 0.080) and anti-receptor binding domain (RBD) IgG (928 [212-3449] BAU/ml vs (83.7 [22.8-2094] BAU/ml, p = 0.066) levels than individuals in the placebo arm (n = 6). Considering individuals who were fully vaccinated with mRNA-based COVID-19 vaccines, the active treatment arm (n = 10) exhibited significantly higher serum levels of anti-RBD IgA (135 [32.9-976] BAU/ml vs 61.3 [26.7-97.1] BAU/ml, p = 0.036) than the placebo arm (n = 7) >28 days postvaccination. Supplementation with specific probiotics might improve the long-term efficacy of mRNA-based COVID-19 vaccines via enhanced IgA response.

Development of a competitive ELISA for NS3 antibodies as DIVA test accompanying the novel Disabled Infectious Single Animal (DISA) vaccine for Bluetongue.

Recently, we have developed a novel vaccine for Bluetongue named BT Disabled Infectious Single Animal (DISA) vaccine. Due to the lack of non-essential NS3/NS3a protein, BT DISA vaccine is a replicating vaccine, but without the inherent risks of live-attenuated vaccines, such as residual virulence or reversion to virulence by mutations, reassortment with field virus, horizontal spread by vectors and vertical transmission. The immune response induced by BT DISA vaccines is rapidly induced, highly protective and serotype specific which is dependent on the immunodominant and serotype determining VP2 protein. The BT DISA vaccine platform provides the replacement of exclusively VP2 from different serotypes in order to safely formulate multivalent cocktail vaccines. The lack of NS3/NS3a directed antibodies by BT DISA vaccination enables differentiation of infected from vaccinated animals (DIVA principle). A highly conserved immunogenic site corresponding to the late domain was mapped in the N-terminal region of NS3. We here established an NS3-specific competitive ELISA (NS3 cELISA) as serological DIVA test accompanying BT DISA vaccines. To this end, NS3 protein missing putative transmembrane regions was produced in large amounts in bacteria and used as antigen in the NS3 cELISA which was investigated with a variety of sera. The NS3 cELISA displayed a high sensitivity and specificity similar to the commercially available VP7-specific cELISA. Results of previously performed vaccination-challenge trials with BT DISA vaccines clearly demonstrate the DIVA system based on the NS3 cELISA and BT vaccine free of NS3 protein.

VP2-serotyped live-attenuated bluetongue virus without NS3/NS3a expression provides serotype-specific protection and enables DIVA.

Bluetongue virus (BTV) causes Bluetongue in ruminants and is transmitted by Culicoides biting midges. Vaccination is the most effective measure to control vector borne diseases; however, there are 26 known BTV serotypes showing little cross protection. The BTV serotype is mainly determined by genome segment 2 encoding the VP2 protein. Currently, inactivated and live-attenuated Bluetongue vaccines are available for a limited number of serotypes, but each of these have their specific disadvantages, including the inability to differentiate infected from vaccinated animals (DIVA). BTV non-structural proteins NS3 and NS3a are not essential for virus replication in vitro, but are important for cytopathogenic effect in mammalian cells and for virus release from insect cells in vitro. Recently, we have shown that virulent BTV8 without NS3/NS3a is non-virulent and viremia in sheep is strongly reduced, whereas local in vivo replication leads to seroconversion. Live-attenuated BTV6 without NS3/NS3a expression protected sheep against BTV challenge. Altogether, NS3/NS3a knockout BTV6 is a promising vaccine candidate and has been named Disabled Infectious Single Animal (DISA) vaccine. Here, we show serotype-specific protection in sheep by DISA vaccine in which only genome segment 2 of serotype 8 was exchanged. Similarly, DISA vaccines against other serotypes could be developed, by exchange of only segment 2, and could therefore safely be combined in multi-serotype cocktail vaccines with respect to reassortment between vaccine viruses. Additionally, NS3 antibody responses are raised after natural BTV infection and NS3-based ELISAs are therefore appropriate tools for DIVA testing accompanying the DISA vaccine. To enable DIVA, we developed an experimental NS3 ELISA. Indeed, vaccinated sheep remained negative for NS3 antibodies, whereas seroconversion for NS3 antibodies was associated with viremia after heterologous BTV challenge.

Homotypic interactions of the infectious bursal disease virus proteins VP3, pVP2, VP4, and VP5: mapping of the interacting domains.

Infectious bursal disease virus (IBDV), a nonenveloped double-stranded RNA virus of chicken, encodes five proteins. Of these, the RNA-dependent RNA polymerase (VP1) is specified by the smaller genome segment, while the large segment directs synthesis of a nonstructural protein (VP5) and a structural protein precursor from which the capsid proteins pVP2 and VP3 as well as the viral protease VP4 are derived. Using the recently redefined processing sites of the precursor, we have reevaluated the homotypic interactions of the viral proteins using the yeast two-hybrid system. Except for VP1, which interacted weakly, all proteins appeared to self-associate strongly. Using a deletion mutagenesis approach, we subsequently mapped the interacting domains in these polypeptides, where possible confirming the observations made in the two-hybrid system by performing coimmunoprecipitation analyses of tagged protein constructs coexpressed in avian culture cells. The results revealed that pVP2 possesses multiple interaction domains, consistent with available structural information about this external capsid protein. VP3-VP3 interactions were mapped to the amino-terminal part of the polypeptide. Interestingly, this domain is distinct from two other interaction domains occurring in this internal capsid protein: while binding to VP1 has been mapped to the carboxy-terminal end of the protein, interaction with the genomic dsRNA segments has been suggested to occur just upstream thereof. No interaction sites could be assigned to the VP4 protein; any deletion applied abolished its self-association. Finally, one interaction domain was detected in the central, most hydrophobic region of VP5, supporting the idea that this virulence determinant may function as a membrane pore-forming protein in infected cells.

Infectious bursal disease virus capsid protein VP3 interacts both with VP1, the RNA-dependent RNA polymerase, and with viral double-stranded RNA.

Infectious bursal disease virus (IBDV) is a double-stranded RNA (dsRNA) virus of the Birnaviridae family. Its two genome segments are encapsidated together with multiple copies of the viral RNA-dependent RNA polymerase, VP1, in a single-shell capsid that is composed of VP2 and VP3. In this study we identified the domains responsible for the interaction between VP3 and VP1. Using the yeast two-hybrid system we found that VP1 binds to VP3 through an internal domain, while VP3 interacts with VP1 solely by its carboxy-terminal 10 amino acids. These results were confirmed by using a reverse-genetics system that allowed us to analyze the interaction of carboxy-terminally truncated VP3 molecules with VP1 in infected cells. Coimmunoprecipitations with VP1- and VP3-specific antibodies revealed that the interaction is extremely sensitive to truncation of VP3. The mere deletion of the C-terminal residue reduced coprecipitation almost completely and also fully abolished production of infectious virions. Surprisingly, these experiments additionally revealed that VP3 also binds to RNA. RNase treatments and reverse transcription-PCR analyses of the immunoprecipitates demonstrated that VP3 interacts with dsRNA of both viral genome segments. This interaction is not mediated by the carboxy-terminal domain of VP3 since C-terminal truncations of 1, 5, or 10 residues did not prevent formation of the VP3-dsRNA complexes. VP3 seems to be the key organizer of birnavirus structure, as it maintains critical interactions with all components of the viral particle: itself, VP2, VP1, and the two genomic dsRNAs.