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.

Live Influenza Vaccine Provides Early Protection against Homologous and Heterologous Influenza and May Prevent Post-Influenza Pneumococcal Infections in Mice.

Influenza and S. pneumoniae infections are a significant cause of morbidity and mortality worldwide. Intranasal live influenza vaccine (LAIV) may prevent influenza-related bacterial complications. The objectives of the study are to estimate resistance against early influenza infection and post-influenza pneumococcal pneumonia after LAIV in mice. Mice were administered intranasally the monovalent LAIV A/17/Mallard Netherlands/00/95(H7N3), A/17/South Africa/2013/01(H1N1)pdm09 or trivalent LAIV 2017-2018 years of formulation containing A/17/New York/15/5364(H1N1)pdm09 vaccine strain. LAIV demonstrated early protection against homologous and heterologous infections with A/South Africa/3626/2013 (H1N1) pdm09 influenza virus on day six, following immunization. Following boost immunization, trivalent LAIV demonstrated a pronounced protective effect both in terms of lethality and pneumococcal lung infection when S. pneumoniae infection was performed three days after the onset of influenza infection. Conclusion: LAIV provides early protection against homologous and heterologous viral infections and has a protective effect against post-influenza pneumococcal infection. These data suggest that the intranasal administration of LAIV may be useful during the cycle of circulation not only of influenza viruses, but also of other causative agents of acute respiratory infections.

Associated virus-bacterial vaccine based on seasonal LAIV and S. pneumoniae chimeric peptide provide protection against post-influenza pneumococcal infection in mouse model.

Severe influenza complications are often caused by Streptococcus pneumoniae infection, which presents the most common cause of community-acquired pneumonia. We evaluated in a mouse model an associated virus-bacterial vaccine based on seasonal live influenza vaccines (LAIV) and S. pneumoniae chimeric protein comprising flagellin (PSPF). Intranasal immunization of mice with a complex of trivalent LAIV and PSPF caused an increased release of early cytokines in the lungs of mice. The immunogenicity of LAIV and PSPF in the associated vaccine composition was sometimes decreased compared to each vaccine preparation alone. Nevertheless, only vaccination of mice with LAIV+PSPF significantly reduced lethality and the bacterial load in the lungs in a model of post-influenza bacterial pneumonia. The study of the interactions of influenza viruses with bacterial peptides is important during the development of associated virus-bacterial vaccines intended for the prevention of severe post-influenza bacterial complications.

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.

Mucosal vaccine based on attenuated influenza virus and the group B Streptococcus recombinant peptides protected mice from influenza and S. pneumoniae infections.

Although many influenza-related deaths are attributable to secondary bacterial infection with S. pneumoniae, vaccines that simultaneously protect against influenza and pneumococcal infection are currently not developed. The aim of our study was to evaluate the possibility to prevent post-influenza pneumococcal infection using an associated vaccine based on live influenza vaccine (LAIV) combined with recombinant polypeptides derived from superficial factors of Group B streptococcus (GBS) determining pathogenicity. We demonstrated in a model of post-influenza pneumococcal pneumonia that intranasal pneumococcal super-infection seriously complicated the course of A/Shanghai/2/2013(H7N9) CDC-RG virus infection in mice. Associated immunization using LAIV and GBS vaccine (GBSV) prevented post-influenza pneumococcal pneumonia better than mono-LAIV or GBSV immunization. At the same time, parenteral pneumococcal post-influenza infection of immune mice was more severe in the groups immunized using recombinant GBS peptides which can be explained by antibody-dependent enhancement of infection. In this case, the introduction of blockers of histamine receptors type 1 and 2 reduced the burden of secondary pneumococcal infection.

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.

Factors of early protective action of live influenza vaccine combined with recombinant bacterial polypeptides against homologous and heterologous influenza infection.

We are developing an associated vaccine based on live influenza vaccine (LAIV) and streptococcal recombinant peptides. The recombinant group B streptococcus (GBS) peptides P6 and ScaAB demonstrated a distinguished immunomodulating effect in THP-1 cells. The increase in IFN 1-alpha expression after ScaAB inoculation was similar to that against LAIV. We immunized mice intranasal using of A/H7N3 LAIV or/and ScaAB peptide. At day 5 after immunization, we detected serum IgM which reacted with non-vaccine influenza viruses. Associated vaccination of mice using LAIV and GBS peptide was the most effective against sub-lethal infection with A/H7N9 influenza virus and against lethal challenge with A/H1N1pdm virus at day 5 after immunization. Not only LAIV but also the ScaAB protected about 20% of the immunized animals against lethal challenge with A/H1N1pdm virus. The early protection was related to increasing type 1 interferons expression in the lungs. Our results in mice have shown that successful protection against homologous and heterologous influenza infections can be achieved soon after vaccination with either LAIV or LAIV in combination with GBS recombinant peptide. Presumably, such protection may be mediated by non-specific IgM antibodies and an increase in the expression of early cytokines in the airway.

Autoprobiotics as an Approach for Restoration of Personalised Microbiota.

Human microbiota is a complex consortium of microorganisms involved in the proper functioning of almost every system of the organism. Majority of the human diseases are associated with the development of intestinal dysbiosis. Dysbiotic condition or dysbiosis is a key pathogenic condition causing many severe infectious or non-infectious diseases. Rapid return to the original microbiota in many cases leads to the fast recovery from the disease. However, the optimal way of the treatment of dysbiosis is still under the discussion. Recently we have developed a method of autoprobiotics based on using isolated indigenous bacteria for improving of microbiota condition. The method based on feeding the patients with bacterial products grown from their personal, genetically characterised strains have been successfully tested in clinic on patients with IBS or chronic pneumonia. In present study we tried to evaluate technology employing autoprobiotic bacteria belonging to different species employing the rat model of antibiotic induced dysbiosis. Six experimental groups of animals after taking antibiotics were treated with different variants of autoprobiotics (lactobacillus, bifidobacteria, enterococcus, their mixture, fecal microbiota, or anaerobically grown complex of indigenous microbiota) prepared for each of them before the development of dysbiosis. Judging by the multiple parameters including metagenomics analysis of microbiota, immune status and microbiota content of the animals with dysbiosis relatively to control group, the most pronounced positive changes were provided by autoprobiotics based on enterococci, bifidobacteria or the consortium of indigenous bacteria grown under anaerobic conditions. These groups of autoprobiotics were delivering the most effective restoration of the original microbiota content and significant anti-inflammatory reaction of the immune system.

Prevention of Influenza A(H7N9) and Bacterial Infections in Mice Using Intranasal Immunization With Live Influenza Vaccine and the Group B Streptococcus Recombinant Polypeptides.

We investigate the protective effect of combined vaccination based on live attenuated influenza vaccine (LAIV) and group B streptococcus (GBS) recombinant polypeptides against potential pandemic H7N9 influenza infection followed by GBS burden. Mice were intranasally immunized using 107 50% egg infectious dose (EID50) of H7N3 LAIV, the mix of the 4 GBS peptides (group B streptococcus vaccine [GBSV]), or combined LAIV + GBSV vaccine. The LAIV raised serum hemagglutination-inhibition antibodies against H7N9 in higher titers than against H7N3. Combined vaccination provided advantageous protection against infections with A/Shanghai/2/2013(H7N9)CDC-RG influenza and serotype II GBS. Combined vaccine significantly improved bacterial clearance from the lungs after infection compared with other vaccine groups. The smallest lung lesions due to combined LAIV + GBSV vaccination were associated with a prevalence of lung interferon-γ messenger RNA expression. Thus, combined viral and bacterial intranasal immunization using H7N3 LAIV and recombinant bacterial polypeptides induced balanced adaptive immune response, providing protection against potential pandemic influenza H7N9 and bacterial complications.

Evaluation in Mouse Model of Combined Virus-bacterial Vaccine Based on Attenuated Influenza A(H7N3) Virus and the Group B Streptococcus Recombinant Polypeptides.

BACKGROUND: Secondary bacterial influenza complications are a common cause of excesses morbidity and mortality, which determines the need to develop means for specific prophylaxis. Group B streptococcal infection is especially common cause of pneumonia among children and the elderly with underlying conditions. Here we investigate in a mouse model the effects of combined intranasal immunization using live attenuated influenza vaccine and recombinant polypeptides based on group B Streptococcus surface proteins. METHODS: Groups of outbred mice received two doses of the following preparations: 1) the reassortant A/17/Mallard/Netherlands/00/95 (H7N3) influenza virus; 2) a mixture of P6, ScaAB, ScpB1 and Stv recombinant GBS proteins (20 µg total); 3) the A(H7N3) influenza vaccine pooled with the four bacterial peptide preparation; 4) control animals were treated with PBS. RESULTS: Intranasal vaccination using LAIV in combination with GBS polypeptides provided advantageous protection against infections with homologous A/Mallard/Netherlands/12/00 (H7N3) wild type virus or heterologous A/Puerto Rico/8/34 (H1N1) followed by serotype II GBS infection. Also, combined vaccination improved bacterial clearance from the lungs of mice. CONCLUSION: Intranasal immunization with LAIV+GBSV was safe and enabled to induce the antibody response to each of vaccine components. Thus, the combined vaccine increased the protective effect against influenza and its bacterial complications in mice compared to LAIV-only.

Central effects of ethanol interact with endogenous mu-opioid activity to control isolation-induced analgesia in maternally separated infant rats.

Endogenous opioid activity plays an important role in ethanol consumption and reinforcement in infant rats. Opioid systems are also involved in mediation and regulation of stress responses. Social isolation is a stressful experience for preweanling rats and changes the effects of ethanol through opioid-dependent mechanisms. The present study assessed effects of intracisternal (i.c.) administration of a selective mu-opioid antagonist (CTOP) and i.p. administration of a nonspecific opioid antagonist (naloxone) on voluntary intake and behavior in socially isolated 12-day-old (P12) pups treated with 0.5 g/kg ethanol. Voluntary intake of 0.1% saccharin or water, locomotion, rearing activity, paw licking and grooming were assessed during short-term isolation from littermates (STSI; 8-min duration). Thermal nociceptive reactivity was measured before and after this intake test, with normalized differences between pre- and post-test latencies of paw withdrawal from a hot plate (49°C) used as an index of isolation-induced analgesia (IIA). Results indicated several effects of social isolation and ethanol mediated through the mu-opioid system. Effects of low dose ethanol (0.5 g/kg) and voluntary consumption of saccharin interacted with endogenous mu-opioid activity associated with STSI. Blockade of mu-opioid receptors on saccharin consumption and paw licking-grooming affected intoxicated animals. Low dose ethanol and ingestion of saccharin blunted effects of CTOP on rearing behavior and nociceptive reactivity. Central injections of CTOP stimulated paw licking and grooming dependent on ethanol dose and type of fluid ingested. Ethanol selectively increased saccharin intake during STSI in females, naloxone and CTOP blocked ethanol-mediated enhancement of saccharin intake. We suggest that enhancement of saccharin intake by ethanol during STSI is the product of synergism between isolation-induced mu-opioid activity that increases the pup's sensitivity to appetitive taste stimulation and the anxiolytic effects of 0.5 g/kg ethanol that decreases behaviors otherwise competing with independent ingestive activity.

µ-Opioid blockade reduces ethanol effects on intake and behavior of the infant rat during short-term but not long-term social isolation.

Numerous findings in adult and infant rats have shown that the endogenous opioid system is involved in control of ethanol consumption and its reinforcing effects. Opioid systems are also involved in reactivity to social isolation with several factors (age, duration, and type of isolation) affecting this modulation. The present study investigated the effects of a selective mu-opioid antagonist CTOP (0, 0.1, 0.5mg/kg), ethanol (0, 0.5 g/kg), and the interaction of the two drugs on the behavioral consequences of two types of social isolation given to preweanling rats: 1) short-term social isolation from littermates (STSI, duration 8 min) and 2) relatively long-term (5h) isolation (LTSI) from the dam and littermates. Voluntary intake of saccharin, locomotion, rearing activity, paw licking, and grooming were assessed during an 8-min. intake test. Thermal nociceptive reactivity was also measured before and after the testing session with normalized differences in pre- and post-test latencies of paw withdrawal from a hot plate (49°C) used as an index of isolation-induced analgesia (IIA). The results indicate that pharmacological blockade of mu-opioid receptors by CTOP substantially attenuated ethanol's anxiolytic effects on the developing rat's reactions to social isolation. Some of these stress-attenuating effects of CTOP were observed only in animals exposed to short-term isolation (STSI) but not in pups isolated for 5h (LTSI). Ethanol selectively increased saccharin intake during STSI in females and CTOP blocked this effect. Ethanol decreased the magnitude of analgesia associated with STSI but had no effect on pain reactivity during LTSI. CTOP by itself did not affect IIA or saccharin intake in sober animals. The findings of the present experiments indicate that the anxiolytic effects of 0.5 g/kg ethanol on pups exposed to STSI are modulated by endogenous opioid activity.

Repetitive exposures to a surrogate nipple providing nutritive and non-nutritive fluids: effects on suckling behavior of the newborn rat.

Responsiveness to a surrogate nipple providing water, 0.1% saccharin, 10% sucrose, pedialyte, or milk was tested in naïve-to-suckling newborn rats during six 10-min exposures, one every 1.5 h over a 7.5 h period. Across a succession of exposures, newborn rats repeatedly attached to and ingested milk from a surrogate nipple, yielding significant body weight gain and increased concentration of blood plasma glucose. Initially, pups ingested considerable amounts of saccharin and sucrose, but then dramatically decreased their consumption of these fluids across the experimental sessions. Intake of milk was significantly higher than that of all other substances. Blood glucose concentration in pups treated with water, saccharin, sucrose, and pedialyte did not differ significantly from that of non-treated pups. The present data suggest a potential contribution of a fluid's palatability and nutritive value in the persistence and efficacy of diet intake for neonatal rats in the context of suckling behavior.