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.

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.

A Live Probiotic Vaccine Prototype Based on Conserved Influenza a Virus Antigens Protect Mice against Lethal Influenza Virus Infection.

BACKGROUND: Due to the highly variable nature of the antigenic properties of the influenza virus, many efforts have been made to develop broadly reactive influenza vaccines. Various vaccine platforms have been explored to deliver conserved viral antigens to the target cells to induce cross-reactive immune responses. Here, we assessed the feasibility of using Enterococcus faecium L3 as a bacterial vector for oral immunization against influenza virus. METHODS: we generated two vaccine prototypes by inserting full-length HA2 (L3-HA2) protein or its long alpha helix (LAH) domain in combination with four M2e tandem repeats (L3-LAH+M2e) into genome of E.faecium L3 probiotic strain. The immunogenicity and protective potential of these oral vaccines were assessed in a lethal challenge model in BALB/c mice. RESULTS: as expected, both vaccine prototypes induced HA stem-targeting antibodies, whereas only L3-LAH+4M2e vaccine induced M2e-specific antibody. The L3-HA2 vaccine partially protected mice against lethal challenge with two H1N1 heterologous viruses, while 100% of animals in the L3-LAH+4M2e vaccine group survived in both challenge experiments, and there was significant protection against weight loss in this group, compared to the L3 vector-immunized control mice. CONCLUSIONS: the recombinant enterococcal strain L3-LAH+4M2e can be considered as a promising live probiotic vaccine candidate for influenza prevention and warrants further evaluation in relevant pre-clinical models.

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.

Development of experimental pneumococcal vaccine for mucosal immunization.

Streptococcus pneumonia is an important human pathogen that causes various severe diseases such as pneumonia, otitis and meningitis. Vaccination against S. pneumoniae is implemented in many developed countries. The presently used vaccines are safe, well tolerated but relatively expensive and require modification due to the immunological changes of the epidemic strains. This paper describes the development of a new pneumococcal vaccine candidate for immunization on mucosal surfaces. For this purpose the antigens of chimeric protein PSPF, previously suggested for an injectable S. pneumoniae vaccine, were expressed on the surface of the live probiotic strain Enterococcus faecium L3. Experiments on laboratory mice vaccinated with live bacteria demonstrated the appearance of the specific IgA and IgG which provide protection against the lethal S. pneumoniae infection.

Experimental poststreptococcal glomerulonephritis elicited by IgG Fc-binding M family proteins and blocked by IgG Fc fragment.

The pathogenesis of acute poststreptococcal glomerulonephritis (APSGN), a major nonsuppurative complication of group A streptococcal (GAS) throat or skin disease, remains unclear. During the years, various theories based on certain streptococcal extracellular factors, as well as immunological mimicry between streptococci and renal tissue, have been forwarded. We earlier reported that many clinical GAS isolates with documented nephritogenic capacity show non-immune binding of monomeric or aggregated IgG. Moreover, in a rabbit model of APSGN we obtained evidence for an important role of streptococcal IgG Fc binding proteins (IgGFcBPs) belonging to the M family surface proteins; thus, hyperimmunization by whole IgGFcBP-positive streptococci was shown to induce renal glomerular changes with deposition of IgG and complement C3, resembling the picture recorded in human APSGN. These typical renal changes were always preceded by the appearance of circulating anti-IgG antibodies. In the present work, using the same rabbit model, each of two purified IgGFcBPs, isolated from type M22 GAS, were found to elicit glomerular degenerative damage comparable to that caused by whole bacteria, as well as formation of anti-IgG. In addition, the induction by whole streptococci (type M1) of experimental APSGN was inhibited by the i.v. administration of purified human or rabbit IgG Fc, but not Fab, fragment, supporting the importance of Fc-mediated mechanisms in causation of glomerulonephritis. We propose that anti-IgG antibody, induced by streptococcal IgGFcBP, facilitated renal accumulation of IgG-containing complexes, which in turn triggered complement deposition and proinflammatory cascades. Further studies on the possible beneficial effect of IgG Fc fragment in APSGN should be of interest.