Elena Bormotova

Background/Objective: This study is a cross-sectional investigation of long-term immune responses measured at different time intervals after COVID-19 infections, vaccinations, or combined exposure. The focus is on immune reactivity against recombinant spike (S) and nucleocapsid (N) protein antigens. Materials and Methods: Serum antibody levels were assessed up to four to four and a half years after infection or immunization, including virus-specific immunoglobulin G (IgG), IgA and IgM antibodies, as well as neutralizing antibodies against the S-protein. Cellular immunity was assessed by analyzing peripheral blood mononuclear cells (PBMC; n = 43 in first cohort, n = 32 in second cohort), including T-helper memory and cytotoxic subsets, and cytokine production after in vitro stimulation with recombinant SARS-CoV-2 proteins. A multiplex cytokine assay was used to analyze effector and regulatory immune responses. Results: Virus-specific IgG antibodies persisted for years after exposure to SARS-CoV-2, with IgG against the receptor-binding domain (RBD) correlating most strongly with neutralizing activity. Vaccinated individuals demonstrated higher IgA responses, whereas antibodies to the N-protein were associated with previous infection. No IgM antibodies were detected in any subjects, suggesting an immune response based on memory rather than ongoing infection. PBMCs from individuals with a history of both COVID-19 exposure and vaccination exhibited enhanced responsiveness, characterized by increased frequencies of memory T cells compared to vaccination alone. Stimulating with the S-protein induces higher cytokine production, including IFN-gamma, TNF-alfa, and IL-12(p70), compared with stimulation by the N-protein. Cytokines such as IL-10 and TGF-beta are also elevated, suggesting immune regulation rather than persistent inflammation. Conclusions: SARS-CoV-2 infection and vaccination are associated with persistent humoral and cellular immune responses detectable several years after exposure. Individuals with hybrid immunity exhibit broader and functionally enhanced immune reactivity, indicating more robust long-term immune memory. Future studies should focus on the long-term consequences of hybrid immunity and optimize other vaccine strategies, including recombinant antigen vaccines.

Background/Objective: This study Investigates long-term immune responses (up to 4–4.5 years) to SARS-CoV-2 in individuals with: Past COVID-19 infection, Vaccination, Combined exposure, with focus on immune reactivity to recombinant spike (S) and nucleocapsid (N) proteins. Materials and methods: Serum antibody responses were assessed up to 4–4.5 years after infection or immunization, including virus-specific IgG, IgA, IgM, and neutralizing antibodies. Cellular immunity was evaluated by phenotypic and functional analysis of peripheral blood mononuclear cells (PBMCs), including memory T-helper and cytotoxic T-cell subsets, as well as cytokine production following in vitro stimulation with recombinant SARS-CoV-2 proteins. Multiplex cytokine profiling was used to characterize effector and regulatory immune responses. Results: Virus-specific IgG antibodies persisted for several years after SARS-CoV-2 exposure, with anti-RBD IgG showing the strongest correlation with virus-neutralizing activity, whereas antibodies to the N- protein primarily reflected prior infection. Vaccinated individuals exhibited a distinct immunoglobulin profile characterized by a higher prevalence of IgA. No IgM detected suggesting the detected immune responses reflect immunological memory rather than active infection. PBMCs from individuals with combined COVID-19 and vaccination history demonstrated enhanced responsiveness and more memory T cells. Hybrid immunity (infection and vaccination) provides stronger and broader immune responses.. Stimulation with S - protein induces stronger cytokine production (IFN-γ, TNF-α, IL-12p70) than N- protein. Regulatory cytokines (IL-10, TGF-β) also elevated which suggests immune regulation rather than chronic inflammation.Conclusion: SARS-CoV-2 infection and vaccination induce persistent humoral and cellular immunity. Neutralizing activity correlates only with anti-RBD and anti-S IgG. Future research should focus on long-term effects, hybrid immunity, and optimizing other vaccine types, in addition to Adenovector vaccines, such as recombinant antigen-based vaccines .

Background: The accumulation of specific IgG antibodies in blood serum is considered a key criterion for the effectiveness of vaccination. For several vaccine-preventable infections, quantitative indicators of the humoral response have been established, which, when reached, provide a high probability of protection against infection. The presence of such a formal correlate of vaccine effectiveness is crucial, for example, in organizing preventive measures and validating newly developed vaccines. However, can effective protection against infection occur when the level of serum antibodies is lower than that provided by parenteral vaccination? Will protection be sufficient if the same vaccine antigen is administered via mucosal membranes without achieving high levels of specific IgG circulating in the blood? Methods: In this study, we compared the immunogenicity and protective efficacy of parenteral and mucosal forms of vaccines in experimental animals, targeting infections caused by the SARS-CoV-2 coronavirus and Streptococcus pneumoniae. We investigated the protective properties of a fragment of the coronavirus S1 protein administered intramuscularly with an adjuvant and orally as part of the probiotic strain Enterococcus faecium L3 in a Syrian hamster model. A comparative assessment of the immunogenicity and protective efficacy of a recombinant tandem (PSP) of immunogenic peptides from S. pneumoniae surface proteins, administered either parenterally or orally, was performed in a Balb/c mouse model. Results: Both models demonstrated significant differences in the immunogenicity of parenteral and oral vaccine antigens, but comparable protective efficacy.

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.

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.

The COVID-19 pandemic was declared in March 2020 and is still ongoing. The course of the epidemic remains uncertain, and mutations in the coronavirus, as well as the emergence of new variants of concern, make it possible that there will be periodic surges in the number of cases. 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 development of pan-coronavirus vaccines that stimulate the production of antibodies against conserved elements of the virus. Another approach could be 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. With a well-established safety profile and the ability to transform into vaccine candidates, probiotic bacteria have significant research potential. 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.

The current pandemic caused by the SARS-Cov-2 virus has significantly influenced the emergence of new injectable vaccines that provide a predominantly specific IgG response. However, it is generally accepted that protection against pathogens at the mucosal surface, which is the first barrier to viral entry, is predominantly dependent on the IgA response. It is now widely accepted that the use of genetically modified microorganisms, including probiotics, allows the oral or nasal mucosal delivery of therapeutic molecules, inducing an immune response in the mucous membranes. Probiotic strains are well studied for safety for the organism and are able to remain viable after passing through the gastric barrier, improve intraepithelial connections, and can generate a number of surface expressed molecules that enhance the effectiveness of vaccination.Recombinant probiotic microorganisms capable of producing vaccine antigens by inserting specific DNA fragments into their genome are one of the potential platforms that can be used to develop an appropriate vaccine containing a specific antigen for rapid response to viral mutations. Here, we demonstrate the construction of a novel SARS-Cov-2 vaccine candidate employing the gene fragment of S1 SARS-Cov-2 gene. According to the available data on new variants of SARS-Cov-2 mutations, three amino acid substitutions were made in the chosen sequence. This DNA fragment was inserted in frame into major pili protein gene within d2 domain of enterococcal operon encoding for pili.

BACKGROUND: Probiotic microorganisms are currently considered as a promising platform for the development of recombinant vaccines expressing viral or bacterial antigens. Probiotic-based mucosal vaccines are easy to produce in large quantities, they have a low cost, provide a fairly long T-cell memory. AIM: The aim was to study expression of mRNA fragment of S1 SARS-CoV-2 gene in Enterococcus faecium L3 culture and to confirm the insertion of S1 SARS-CoV-2 protein fragment into the pili of this bacterial strain by immunoelectron microscopy of original (E. faecium L3) and genetically modified strain (L3-SARS) with human sera obtained from patients with SARS-CoV-2. MATERIALS AND METHODS: mRNA expression was studied by real-time PCR with reverse transcription using primers specific to S1 protein. Immunoelectron microscopy was aimed to study the structure of E. faecium L3 pili with the expression of viral protein SARS-CoV-2. Bacteria were washed three times in PBS by centrifugation at 3500 rpm for 20 min and suspended in 0.1 M NaCl. A 10-fold bacterial concentrate was used. The source of the primary antibodies was a set of polyclonal human sera containing IgG. Labeling was performed using goat IgG conjugated with 18 nm gold particles. RESULTS: A sharp increase in mRNA amplification of inserted genetic sequence of S1 SARS-CoV-2 gene fragment relatively to the control was demonstrated. These results confirmed that DNA of S1 gene in E. faecium L3 genome is transcribed together with the target pili gene in E. faecium genome. Specific antigens of SARS-CoV-2 on the surface of L3-SARS were determined using electron microscopy, which demonstrated the correct assembly of chimeric molecules of pili on the surface of bacteria. CONCLUSIONS: Evaluation in expression of SARS-CoV-2 S1 protein after introduction of the corresponding genetic elements into genome of probiotic strain E. faecium L3 allows us to conclude that selected DNA fragments of SARS-CoV-2 were able to direct the synthesis of immunogenic protein S1 that was expressed by the strain E. faecium L3-SARS.

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.

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.