SARS-CoV-2 Spike Protein-Expressing Enterococcus for Oral Vaccination: Immunogenicity and Protection.

The declaration of the conclusion of the COVID-19 pandemic notwithstanding, coronavirus remains prevalent in circulation, and the potential emergence of novel variants of concern introduces the possibility of new outbreaks. Moreover, it is not clear how quickly and to what extent the effectiveness of vaccination will decline as the virus continues to mutate. One possible solution to combat the rapidly mutating coronavirus is the creation of safe vaccine platforms that can be rapidly adapted to deliver new, specific antigens in response to viral mutations. Recombinant probiotic microorganisms that can produce viral antigens by inserting specific viral DNA fragments into their genome show promise as a platform and vector for mucosal vaccine antigen delivery. The authors of this study have developed a convenient and universal technique for inserting the DNA sequences of pathogenic bacteria and viruses into the gene that encodes the pili protein of the probiotic strain E. faecium L3. The paper presents data on the immunogenic properties of two E. faecium L3 vaccine strains, which produce two different fragments of the coronavirus S1 protein, and provides an assessment of the protective efficacy of these oral vaccines against coronavirus infection in Syrian hamsters.

Pilot Study Results on Antibodies to the S- and N-Proteins of SARS-CoV-2 in Paired Sera from COVID-19 Patients with Varying Severity.

In this retrospective cohort study, we investigated the formation of individual classes of antibodies to SARS-CoV-2 in archived serial sera from hospitalized patients with the medium-severe (n = 17) and severe COVID-19 (n = 11). The serum/plasma samples were studied for the presence of IgG, IgM and IgA antibodies to the recombinant S- and N-proteins of SARS-CoV-2. By the 7th day of hospitalization, an IgG increase was observed in patients both with a positive PCR test and without PCR confirmation of SARS-CoV-2 infection. Significant increases in the anti-spike IgG levels were noted only in moderate COVID-19. The four-fold increase of IgM to N-protein was obtained more often in the groups with mild and moderate infections. The IgA levels decreased during the infection to both the S- and N-proteins, and the most pronounced decrease was in the severe COVID-19 patients. The serum IgG to S-protein one week after hospitalization demonstrated a high-power relationship (rs = 0.75) with the level of RBD antibodies. There was a medium strength relationship between the levels of CRP and IgG (rs = 0.43). Thus, in patients with acute COVID-19, an increase in antibodies can develop as early as 1 week of hospital stay. The SARS-CoV-2 antibody conversions may confirm SARS-CoV-2 infection in PCR-negative patients.

Evaluation of Immune Response to Mucosal Immunization with an Oral Probiotic-Based Vaccine in Mice: Potential for Prime-Boost Immunization against SARS-CoV-2.

Following the conclusion of the COVID-19 pandemic, the persistent genetic variability in the virus and its ongoing circulation within the global population necessitate the enhancement of existing preventive vaccines and the development of novel ones. A while back, we engineered an orally administered probiotic-based vaccine, L3-SARS, by integrating a gene fragment that encodes the spike protein S of the SARS-CoV-2 virus into the genome of the probiotic strain E. faecium L3, inducing the expression of viral antigen on the surface of bacteria. Previous studies demonstrated the efficacy of this vaccine candidate in providing protection against the virus in Syrian hamsters. In this present study, utilizing laboratory mice, we assess the immune response subsequent to immunization via the gastrointestinal mucosa and discuss its potential as an initial phase in a two-stage vaccination strategy. Our findings indicate that the oral administration of L3-SARS elicits an adaptive immune response in mice. Pre-immunization with L3-SARS enhances and prolongs the humoral immune response following a single subcutaneous immunization with a recombinant S-protein analogous to the S-insert of the coronavirus in Enterococcus faecium L3.

Developing a Live Probiotic Vaccine Based on the Enterococcus faecium L3 Strain Expressing Influenza Neuraminidase.

Probiotic microorganisms are currently considered as a promising platform for the development of recombinant vaccines expressing foreign antigens. In this study, we generated and evaluated the live mucosal recombinant vaccine by integrating genes encoding influenza virus neuraminidase (NA) of the N2 subtype into the DNA of the probiotic strain Enterococcus faecium L3 (L3). We confirmed NA expression in the pili of L3 using immune electron microscopy. Mice were fed with a probiotic vaccine containing the NA gene (L3-NA) or pure L3. Oral administration of L3-NA caused detectable increase in virus-specific serum IgG and local IgA after the third feeding. Immunization with L3-NA increased the survival rate by 34% when the mice were infected using A(H1N1)pdm09 influenza virus after the third feeding. After S. pneumoniae post-influenza infection, the L3-NA-immunized mice were 50% more protected from lethality in comparison with L3-fed mice. Thus, a live probiotic vaccine candidate based on L3 induced the formation of systemic and local immunity and provide partial protection against complicated influenza.

Construction of the Enterococcal Strain Expressing Immunogenic Fragment of SARS-Cov-2 Virus.

Contemporary SARS-Cov-2 pandemic, besides its dramatic global influence on the human race including health care systems, economies, and political decisions, opened a window for the global experiment with human vaccination employing novel injectable vaccines providing predominantly specific IgG response with little knowledge of their impact on the mucosal immunity. However, it is widely accepted that protection against the pathogens at the gates of the infection - on mucosal surfaces-predominantly rely on an IgA response. Some genetically modified bacteria, including probiotics, represent attractive vehicles for oral or nasal mucosal delivery of therapeutic molecules. Probiotic-based vaccines for mucous membranes are easy to produce in large quantities; they have low cost, provide quite a long T-cell memory, and gut IgA response to oral vaccines is highly synchronized and strongly oligoclonal. Here we present a study demonstrating construction of the novel SARS-Cov-2 vaccine candidate employing the gene fragment of S1 SARS-Cov-2 gene. This DNA fragment was inserted in frame into major pili protein gene with d2 domain of enterococcal operon encoding for pili. The DNA sequencing proved the presence of the insert in enterococcal genome. RNA transcription, immunoprecipitation, and immune electron microscopy with human sera obtained from the SARS-Cov-2 patients demonstrated expression of SARS-Cov-2 antigens in bacteria. Taken together the data obtained allowed considering this genetically modified probiotic strain as an interesting candidate for vaccine against SARS-Cov-2.