Surface immunogenic protein from Streptococcus agalactiae and Fissurella latimarginata hemocyanin are TLR4 ligands and activate MyD88- and TRIF dependent signaling pathways.

The development of vaccine adjuvants is of interest for the management of chronic diseases, cancer, and future pandemics. Therefore, the role of Toll-like receptors (TLRs) in the effects of vaccine adjuvants has been investigated. TLR4 ligand-based adjuvants are the most frequently used adjuvants for human vaccines. Among TLR family members, TLR4 has unique dual signaling capabilities due to the recruitment of two adapter proteins, myeloid differentiation marker 88 (MyD88) and interferon-ß adapter inducer containing the toll-interleukin-1 receptor (TIR) domain (TRIF). MyD88-mediated signaling triggers a proinflammatory innate immune response, while TRIF-mediated signaling leads to an adaptive immune response. Most studies have used lipopolysaccharide-based ligands as TLR4 ligand-based adjuvants; however, although protein-based ligands have been proven advantageous as adjuvants, their mechanisms of action, including their ability to undergo structural modifications to achieve optimal immunogenicity, have been explored less thoroughly. In this work, we characterized the effects of two protein-based adjuvants (PBAs) on TLR4 signaling via the recruitment of MyD88 and TRIF. As models of TLR4-PBAs, we used hemocyanin from Fissurella latimarginata (FLH) and a recombinant surface immunogenic protein (rSIP) from Streptococcus agalactiae. We determined that rSIP and FLH are partial TLR4 agonists, and depending on the protein agonist used, TLR4 has a unique bias toward the TRIF or MyD88 pathway. Furthermore, when characterizing gene products with MyD88 and TRIF pathway-dependent expression, differences in TLR4-associated signaling were observed. rSIP and FLH require MyD88 and TRIF to activate nuclear factor kappa beta (NF-κB) and interferon regulatory factor (IRF). However, rSIP and FLH have a specific pattern of interleukin 6 (IL-6) and interferon gamma-induced protein 10 (IP-10) secretion associated with MyD88 and TRIF recruitment. Functionally, rSIP and FLH promote antigen cross-presentation in a manner dependent on TLR4, MyD88 and TRIF signaling. However, FLH activates a specific TRIF-dependent signaling pathway associated with cytokine expression and a pathway dependent on MyD88 and TRIF recruitment for antigen cross-presentation. Finally, this work supports the use of these TLR4-PBAs as clinically useful vaccine adjuvants that selectively activate TRIF- and MyD88-dependent signaling to drive safe innate immune responses and vigorous Th1 adaptive immune responses.

Humoral immunity against SARS-CoV-2 evoked by heterologous vaccination groups using the CoronaVac (Sinovac) and BNT162b2 (Pfizer/BioNTech) vaccines in Chile.

Introduction: Severe acute respiratory syndrome virus 2 (SARS-CoV-2) has caused over million deaths worldwide, with more than 61,000 deaths in Chile. The Chilean government has implemented a vaccination program against SARS-CoV-2, with over 17.7 million people receiving a complete vaccination scheme. The final target is 18 million individuals. The most common vaccines used in Chile are CoronaVac (Sinovac) and BNT162b2 (Pfizer-Biotech). Given the global need for vaccine boosters to combat the impact of emerging virus variants, studying the immune response to SARS-CoV-2 is crucial. In this study, we characterize the humoral immune response in inoculated volunteers from Chile who received vaccination schemes consisting of two doses of CoronaVac [CoronaVac (2x)], two doses of CoronaVac plus one dose of BNT162b2 [CoronaVac (2x) + BNT162b2 (1x)], and three doses of BNT162b2 [BNT162b2 (3x)]. Methods: We recruited 469 participants from Clínica Dávila in Santiago and the Health Center Víctor Manuel Fernández in the city of Concepción, Chile. Additionally, we included participants who had recovered from COVID-19 but were not vaccinated (RCN). We analyzed antibodies, including anti-N, anti-S1-RBD, and neutralizing antibodies against SARS-CoV-2. Results: We found that antibodies against the SARS-CoV-2 nucleoprotein were significantly higher in the CoronaVac (2x) and RCN groups compared to the CoronaVac (2x) + BNT162b2 (1x) or BNT162b2 (3x) groups. However, the CoronaVac (2x) + BNT162b2 (1x) and BNT162b2 (3x) groups exhibited a higher concentration of S1-RBD antibodies than the CoronaVac (2x) group and RCN group. There were no significant differences in S1-RBD antibody titers between the CoronaVac (2x) + BNT162b2 (1x) and BNT162b2 (3x) groups. Finally, the group immunized with BNT162b2 (3x) had higher levels of neutralizing antibodies compared to the RCN group, as well as the CoronaVac (2x) and CoronaVac (2x) + BNT162b2 (1x) groups. Discussion: These findings suggest that vaccination induces the secretion of antibodies against SARS-CoV-2, and a booster dose of BNT162b2 is necessary to generate a protective immune response. In the current state of the pandemic, these data support the Ministry of Health of the Government of Chile's decision to promote heterologous vaccination as they indicate that a significant portion of the Chilean population has neutralizing antibodies against SARS-CoV-2.

Protein-Based Adjuvants for Vaccines as Immunomodulators of the Innate and Adaptive Immune Response: Current Knowledge, Challenges, and Future Opportunities.

New-generation vaccines, formulated with subunits or nucleic acids, are less immunogenic than classical vaccines formulated with live-attenuated or inactivated pathogens. This difference has led to an intensified search for additional potent vaccine adjuvants that meet safety and efficacy criteria and confer long-term protection. This review provides an overview of protein-based adjuvants (PBAs) obtained from different organisms, including bacteria, mollusks, plants, and humans. Notably, despite structural differences, all PBAs show significant immunostimulatory properties, eliciting B-cell- and T-cell-mediated immune responses to administered antigens, providing advantages over many currently adopted adjuvant approaches. Furthermore, PBAs are natural biocompatible and biodegradable substances that induce minimal reactogenicity and toxicity and interact with innate immune receptors, enhancing their endocytosis and modulating subsequent adaptive immune responses. We propose that PBAs can contribute to the development of vaccines against complex pathogens, including intracellular pathogens such as Mycobacterium tuberculosis, those with complex life cycles such as Plasmodium falciparum, those that induce host immune dysfunction such as HIV, those that target immunocompromised individuals such as fungi, those with a latent disease phase such as Herpes, those that are antigenically variable such as SARS-CoV-2 and those that undergo continuous evolution, to reduce the likelihood of outbreaks.

Validation of a Methodology for the Detection of Severe Acute Respiratory Syndrome Coronavirus 2 in Saliva by Real-Time Reverse Transcriptase-PCR.

In January 2021, the Chilean city of Concepción experienced a second wave of coronavirus 2019 (COVID-19) while in early April 2021, the entire country faced the same situation. This outbreak generated the need to modify and validate a method for detecting severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in saliva, thereby expanding the capacity and versatility of testing for COVID-19. This study was conducted in February 2021 in the Chilean city of Concepción during which time, the town was under total quarantine. The study participants were mostly symptomatic (87.4%), not hospitalized, and attended care centers because of their health status rather than being asked by the researchers. People coming to the health center in Concepción to be tested for COVID-19 (via reverse transcriptase polymerase chain reaction [RT-PCR]) from a specimen of nasopharyngeal swab (NPS) were then invited to participate in this study. A total of 131 participants agreed to sign an informed consent and to provide saliva and NPS specimens to validate a method in terms of sensitivity, specificity, and statistical analysis of the cycle threshold (Ct) values from the RT-PCR. Calculations pertaining to the 127 participants who were ultimately included in the analysis showed sensitivity and specificity at 94.34% (95% CI: 84.34-98.82%) and 98.65% (95% CI: 92.70-99.97%), respectively. The saliva specimen showed a performance comparable to NPS as demonstrated by the diagnostic parameters. This RT-PCR method from the saliva specimen is a highly sensitive and specific alternative compared to the reference methodology, which uses the NPS specimen. This modified and validated method is intended for use in the in vitro diagnosis of SARS-CoV-2, which provides health authorities in Chile and local laboratories with a real testing alternative to RT-PCR from NPS.

Host Components That Modulate the Disease Caused by hMPV.

Human metapneumovirus (hMPV) is one of the main pathogens responsible for acute respiratory infections in children up to 5 years of age, contributing substantially to health burden. The worldwide economic and social impact of this virus is significant and must be addressed. The structural components of hMPV (either proteins or genetic material) can be detected by several receptors expressed by host cells through the engagement of pattern recognition receptors. The recognition of the structural components of hMPV can promote the signaling of the immune response to clear the infection, leading to the activation of several pathways, such as those related to the interferon response. Even so, several intrinsic factors are capable of modulating the immune response or directly inhibiting the replication of hMPV. This article will discuss the current knowledge regarding the innate and adaptive immune response during hMPV infections. Accordingly, the host intrinsic components capable of modulating the immune response and the elements capable of restricting viral replication during hMPV infections will be examined.

Development and analytical validation of real-time PCR for the detection of Streptococcus agalactiae in pregnant women.

BACKGROUND: Group B Streptococcus (GBS) is the leading cause of invasive neonatal infection. In this study, we aimed to evaluate the analytical validation of qualitative real-time polymerase chain reaction (qPCR) as a means to detect GBS. METHODS: Genomic DNA (gDNA) was purified from 12 ATCC bacterial strains, two belonging to GBS and the remainder acting as negative controls. Additionally, gDNA was isolated from 21 strains of GBS from various serotypes (Ia, Ib and II-VIII). All gDNA was used to evaluate the analytical validation of the qPCR method employing a specific Taqman probe. Inclusivity, exclusivity, anticipated reportable range, the limit of detection and robustness were evaluated. The methods used are described in international guidelines and other existing reports. The performance of this qPCR method for detecting GBS was compared to other microbiological methods used with vaginal-rectal samples from pregnant women. RESULTS: Our qPCR method for detecting GBS was analytically validated. It has a limit of detection of 0.7 GE/µL and 100% analytical specificity. It detects all strains of GBS with the same level of performance as microbiological methods. CONCLUSION: Data suggest that this qPCR method performs adequately as a means to detect GBS in vaginal-rectal swabs from pregnant women.

Mucosal Vaccination with Lactococcus lactis-Secreting Surface Immunological Protein Induces Humoral and Cellular Immune Protection against Group B Streptococcus in a Murine Model.

Group B Streptococcus (GBS) is the primary etiological agent of sepsis and meningitis in newborns and is associated with premature birth and stillbirth. The development of a licensed vaccine is one of the pending challenges for the World Health Organization. Previously, we showed that oral immunization with surface immune protein (SIP) decreases vaginal colonization of GBS and generates functional opsonizing antibodies, which was determined by opsonophagocytic assays (OPA) in vitro. We also showed that the protein has an adjuvant vaccine profile. Therefore, an oral vaccine based on SIP may be an attractive alternative to employ in the development of new vaccines against GBS. Lactococcus lactis is a highlighted oral vaccine probiotic inducer of the mucosal immune response. This bacterium could serve as an antigen-delivering vehicle for the development of an edible vaccine and has been used in clinical trials. In this study, we showed that an oral vaccine with a recombinant L. lactis strain secreting SIP from GBS (rL. lactis-SIP) can induce protective humoral and cellular immunity in an experimental model of GBS vaginal colonization in C57BL/6 mice. Mice immunized with rL. lactis-SIP were protected against clinical symptoms and bacterial colonization after GBS vaginal colonization. Our rL. lactis-SIP vaccine also induces an increase of immunoglobulin G (IgG) and immunoglobulin A (IgA) specifically against SIP. The adoptive transfer of serum from vaccinated mice to naïve mice generated protection against GBS vaginal colonization. Moreover, the rL. lactis-SIP strain induces the activation of SIP-specific T cells, which could decrease GBS vaginal colonization and generate protective antibodies when transferred to other mice. Our experimental observations strongly support the notion that rL. lactis-SIP induces protective humoral and cellular immunity and could be considered as a novel alternative in the development of vaccines for GBS.

Surface Immunogenic Protein of Streptococcus Group B is an Agonist of Toll-Like Receptors 2 and 4 and a Potential Immune Adjuvant.

Vaccine-induced protection against pathogens, especially subunit-based vaccines, are related to antigen properties but mainly in their ability to stimulate the immune system by the use of an adjuvant. Modern vaccines are formulated with a high level of antigen purity, where an efficient adjuvant is necessary. In this context, the use of protein Toll-Like Receptor (TLR) agonists as vaccine adjuvants has been highlighted because of their optimal immunogenicity and minimal toxicity. The Surface Immunogenic Protein (SIP) from Group B Streptococcus (GBS) has gained importance as a new potential protein-based vaccine. Recently, we reported that recombinant SIP (rSIP) expressed by E. coli and purified by High Performance Liquid Chromatography (HPLC) alone induces a protective humoral immune response. In this study, we present the immunomodulatory properties of rSIP as a protein-based adjuvant, as an agonist of TLR. To this end, we showed that C57BL/6 bone marrow-derived dendritic cells pulsed by rSIP resulted in enhanced CD40, CD80, CD86, and Major Histocompatibility Complex (MHC) class II as well as increased secretion proinflammatory cytokines Interleukin (IL)-6, Interferon (IFN)-γ, Tumor Necrosis Factor (TNF)-α, and IL-10. Next, we investigated the in vivo effect of rSIP in the absence or presence of ovalbumin (OVA) on antigen-specific antibody secretion in C57BL/6 mice. Immunization with rSIP plus OVA showed that anti-OVA IgG2a and IgG1a increased significantly compared with OVA alone in C57BL/6 mice. Also, the immunization of rSIP plus OVA generates increased serum cytokines levels characterized by IL-12p70, IL-10, IL-4, and IFN-γ. Interestingly, we observed that rSIP stimulate Toll Like Receptor (TLR)2 and TLR4, individually expressed by Human embryonic kidney (HEK) 293-derived TLR reporter cells. These findings suggest that rSIP is a new potential protein TLR agonist adjuvant and may be employed in the development of new vaccines.

BCG-Induced Cross-Protection and Development of Trained Immunity: Implication for Vaccine Design.

The Bacillus Calmette-Guérin (BCG) is a live attenuated tuberculosis vaccine that has the ability to induce non-specific cross-protection against pathogens that might be unrelated to the target disease. Vaccination with BCG reduces mortality in newborns and induces an improved innate immune response against microorganisms other than Mycobacterium tuberculosis, such as Candida albicans and Staphylococcus aureus. Innate immune cells, including monocytes and natural killer (NK) cells, contribute to this non-specific immune protection in a way that is independent of memory T or B cells. This phenomenon associated with a memory-like response in innate immune cells is known as "trained immunity." Epigenetic reprogramming through histone modification in the regulatory elements of particular genes has been reported as one of the mechanisms associated with the induction of trained immunity in both, humans and mice. Indeed, it has been shown that BCG vaccination induces changes in the methylation pattern of histones associated with specific genes in circulating monocytes leading to a "trained" state. Importantly, these modifications can lead to the expression and/or repression of genes that are related to increased protection against secondary infections after vaccination, with improved pathogen recognition and faster inflammatory responses. In this review, we discuss BCG-induced cross-protection and acquisition of trained immunity and potential heterologous effects of recombinant BCG vaccines.

Cellular immune response induced by surface immunogenic protein with AbISCO-100 adjuvant vaccination decreases group B Streptococcus vaginal colonization.

Group B Streptococcus (GBS) represents one of the most common causes of bacterial infection in neonates; it is also associated with premature childbirth and stillbirth. A vaccine against GBS is needed, but no approved vaccines are yet available. The Surface Immunogenic Protein (SIP) of GBS is conserved in all serotypes and had been reported to be a good vaccine prototype in a mouse model of GBS infection. Also, we have previously shown that both subcutaneous and oral immunization with rSIP can induce an efficient immune response that decreases GBS vaginal colonization in mice. In this study, we show that a vaccine based on a mixture of rSIP and AbISCO-100 adjuvant reduces GBS vaginal colonization in mice and induces antibodies with opsonophagocytic activities. Moreover, the passive transfer of sera and total T-cells from mice immunized with rSIP mixed with AbISCO-100 to unvaccinated mice decreases vaginal GBS colonization in an infected mouse. This is the first report of cellular immunity associated with rSIP-based vaccine testing in a mouse model of GBS infection.

Current Animal Models for Understanding the Pathology Caused by the Respiratory Syncytial Virus.

The human respiratory syncytial virus (hRSV) is the main etiologic agent of severe lower respiratory tract infections that affect young children throughout the world, associated with significant morbidity and mortality, becoming a serious public health problem globally. Up to date, no licensed vaccines are available to prevent severe hRSV-induced disease, and the generation of safe-effective vaccines has been a challenging task, requiring constant biomedical research aimed to overcome this ailment. Among the difficulties presented by the study of this pathogen, it arises the fact that there is no single animal model that resembles all aspects of the human pathology, which is due to the specificity that this pathogen has for the human host. Thus, for the study of hRSV, different animal models might be employed, depending on the goal of the study. Of all the existing models, the murine model has been the most frequent model of choice for biomedical studies worldwide and has been of great importance at contributing to the development and understanding of vaccines and therapies against hRSV. The most notable use of the murine model is that it is very useful as a first approach in the development of vaccines or therapies such as monoclonal antibodies, suggesting in this way the direction that research could have in other preclinical models that have higher maintenance costs and more complex requirements in its management. However, several additional different models for studying hRSV, such as other rodents, mustelids, ruminants, and non-human primates, have been explored, offering advantages over the murine model. In this review, we discuss the various applications of animal models to the study of hRSV-induced disease and the advantages and disadvantages of each model, highlighting the potential of each model to elucidate different features of the pathology caused by the hRSV infection.

The Optimisation of the Expression of Recombinant Surface Immunogenic Protein of Group B Streptococcus in Escherichia coli by Response Surface Methodology Improves Humoral Immunity.

Group B Streptococcus (GBS) is the leading cause of neonatal meningitis and a common pathogen in livestock and aquaculture industries around the world. Conjugate polysaccharide and protein-based vaccines are under development. The surface immunogenic protein (SIP) is a conserved protein in all GBS serotypes and has been shown to be a good target for vaccine development. The expression of recombinant proteins in Escherichia coli cells has been shown to be useful in the development of vaccines, and the protein purification is a factor affecting their immunogenicity. The response surface methodology (RSM) and Box-Behnken design can optimise the performance in the expression of recombinant proteins. However, the biological effect in mice immunised with an immunogenic protein that is optimised by RSM and purified by low-affinity chromatography is unknown. In this study, we used RSM for the optimisation of the expression of the rSIP, and we evaluated the SIP-specific humoral response and the property to decrease the GBS colonisation in the vaginal tract in female mice. It was observed by NI-NTA chromatography that the RSM increases the yield in the expression of rSIP, generating a better purification process. This improvement in rSIP purification suggests a better induction of IgG anti-SIP immune response and a positive effect in the decreased GBS intravaginal colonisation. The RSM applied to optimise the expression of recombinant proteins with immunogenic capacity is an interesting alternative in the evaluation of vaccines in preclinical phase, which could improve their immune response.

Immunization with a Mixture of Nucleoprotein from Human Metapneumovirus and AbISCO-100 Adjuvant Reduces Viral Infection in Mice Model.

The human metapneumovirus (hMPV) is the second leading cause globally of acute infection of the respiratory tract in children, infecting the upper and lower airways. The hMPV may induce an inappropriate Th2-type immune response, which causes severe pulmonary inflammation, leading to the obstruction of airways. Despite its severe epidemiological relevance, no vaccines are currently available for the prevention of hMPV-induced illness. In this investigation, we demonstrated that immunization of mice with the recombinant hMPV nucleoprotein (hMPV-N) mixed with the AbISCO-100 adjuvant reduced viral replication in lungs following challenge with the virus. We found that immunized mice had reduced weight loss, decreased granulocytes in the lung, an increased level of specific nucleoprotein antibodies of IgG1 and IgG2a-isotypes, and a local profile of Th1/Th17-type cytokines. Our results suggest that immunization with the hMPV-N and the AbISCO-100 adjuvant induces a reduction of viral infection and could be considered for the development of an hMPV vaccine.

Oral administration of recombinant Neisseria meningitidis PorA genetically fused to H. pylori HpaA antigen increases antibody levels in mouse serum, suggesting that PorA behaves as a putative adjuvant.

The Neisseria meningitidis outer membrane protein PorA from a Chilean strain was purified as a recombinant protein. PorA mixed with AbISCO induced bactericidal antibodies against N. meningitidis in mice. When PorA was fused to the Helicobacter pylori HpaA antigen gene, the specific response against H. pylori protein increased. Splenocytes from PorA-immunized mice were stimulated with PorA, and an increase in the secretion of IL-4 was observed compared with that of IFN-γ. Moreover, in an immunoglobulin sub-typing analysis, a substantially higher IgG1 level was found compared with IgG2a levels, suggesting a Th2-type immune response. This study revealed a peculiar behavior of the purified recombinant PorA protein per se in the absence of AbISCO as an adjuvant. Therefore, the resistance of PorA to proteolytic enzymes, such as those in the gastrointestinal tract, was analyzed, because this is an important feature for an oral protein adjuvant. Finally, we found that PorA fused to the H. pylori HpaA antigen, when expressed in Lactococcus lactis and administered orally, could enhance the antibody response against the HpaA antigen approximately 3 fold. These observations strongly suggest that PorA behaves as an effective oral adjuvant.

Local cytokine response upon respiratory syncytial virus infection.

Respiratory syncytial virus (RSV) is the leading cause of childhood hospitalization and respiratory distress and has been recognized for several decades as a major health and economic burden worldwide. This virus has developed several virulence mechanisms to impair the establishment of a protective immune response to re-infection. Accordingly, inefficient immunological memory is usually generated after exposure to this pathogen. Furthermore, it has been shown that RSV can actively promote the induction of an inadequate cellular immune response at the site of infection that causes exacerbated inflammation in the respiratory tract. Such an inflammatory response is both inefficient for clearing the virus and can be responsible for detrimental symptoms, such as asthma and wheezing. Recent data suggest that RSV possesses molecular mechanisms to induce the secretion of pro-inflammatory cytokines that modulate the immune response and impair viral clearance by reducing IFN-γ production. Here, we discuss recent research leading to the identification of RSV virulence factors that are responsible of promoting a pro-inflammatory environment at the airways and their implications on pathogenicity.