Immune Responses to Orally Administered Recombinant Lactococcus lactis Expressing Multi-Epitope Proteins Targeting M Cells of Foot-and-Mouth Disease Virus

Foot and mouth disease virus (FMDV), whose transmission occurs through mucosal surfaces, can also be transmitted through aerosols, direct contact, and pollutants. Therefore, mucosal immunity can efficiently inhibit viral colonization. Since vaccine material delivery into immune sites is important for efficient oral mucosal vaccination, the M cell-targeting approach is important for effective vaccination given M cells are vital for luminal antigen influx into the mucosal lymph tissues. In this study, we coupled M cell-targeting ligand Co1 to multi-epitope TB1 of FMDV to obtain TB1-Co1 in order to improve delivery efficiency of the multi-epitope protein antigen TB1. Lactococcus lactis (L. lactis) was engineered to express heterologous antigens for applications as vaccine vehicles with the ability to elicit mucosal as well as systemic immune responses. We successfully constructed L. lactis (recombinant) with the ability to express multi-epitope antigen proteins (TB1 and TB1-Co1) of the FMDV serotype A (named L. lactis-TB1 and L. lactis-TB1-Co1). Then, we investigated the immunogenic potential of the constructed recombinant L. lactis in mice and guinea pigs. Orally administered L. lactis-TB1 as well as L. lactis-TB1-Co1 in mice effectively induced mucosal secretory IgA (SIgA) and IgG secretion, development of a strong cell-mediated immune reactions, substantial T lymphocyte proliferation in the spleen, and upregulated IL-2, IFN-γ, IL-10, and IL-5 levels. Orally administered ligand-conjugated TB1 promoted specific IgG as well as SIgA responses in systemic and mucosal surfaces, respectively, when compared to orally administered TB1 alone. Then, guinea pigs were orally vaccinated with L. lactis-TB1-Co1 plus adjuvant CpG-ODN at three different doses, L. lactis-TB1-Co1, and PBS. Animals that had been immunized with L. lactis-TB1-Co1 plus adjuvant CpG-ODN and L. lactis-TB1-Co1 developed elevated antigen-specific serum IgG, IgA, neutralizing antibody, and mucosal SIgA levels, when compared to control groups. Particularly, in mice, L. lactis-TB1-Co1 exhibited excellent immune effects than L. lactis-TB1. Therefore, L. lactis-TB1-Co1 can induce elevations in mucosal as well as systemic immune reactions, and to a certain extent, provide protection against FMDV. In conclusion, M cell-targeting approaches can be employed in the development of effective oral mucosa vaccines for FMDV.     

Dendritic cell targeting of Bacillus anthracis protective antigen expressed by Lactobacillus acidophilus protects mice from lethal challenge

Efficient vaccines potentiate antibody avidity and increase T cell longevity, which confer protection against microbial lethal challenge. A vaccine strategy was established by using Lactobacillus acidophilus to deliver Bacillus anthracis protective antigen (PA) via specific dendritic cell-targeting peptides to dendritic cells (DCs), which reside in the periphery and mucosal surfaces, thus directing and regulating acquired immunity. The efficiency of oral delivery of L. acidophilus expressing a PA-DCpep fusion was evaluated in mice challenged with lethal B. anthracis Sterne. Vaccination with L. acidophilus expressing PA-DCpep induced robust protective immunity against B. anthracis Sterne compared with mice vaccinated with L. acidophilus expressing PA-control peptide or an empty vector. Additionally, serum anti-PA titers, neutralizing PA antibodies, and the levels of IgA-expressing cells were all comparable with the historical recombinant PA plus aluminum hydroxide vaccine administered s.c. Collectively, development of this strategy for oral delivery of DC-targeted antigens provides a safe and protective vaccine via a bacterial adjuvant that may potentiate mucosal immune responses against deadly pathogens.     

Neutralization of SARS-CoV-2 Variants by Serum from BNT162b2 Vaccine Recipients

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has evolved rapidly, leading to viral lineages characterized by multiple mutations in the spike protein, which could potentially confer to the virus the ability to avoid the vaccine-induced immune response, making the vaccines less effective or ineffective. Here, we initially evaluated the neutralization capabilities in vitro by serum neutralization (SN) of six serum samples collected from recipients of the BNT162b2 vaccine against 11 SARS-CoV-2 isolates belonging to the major SARS-CoV-2 lineages that had been circulating in Italy. Then, we considered 30 additional serum samples by SN assay against the dominant B.1.617.2 (Delta) variant. A B.1 lineage isolate was used as a reference. In the first analysis, significant differences when compared with the reference strain (p > 0.05) were not evidenced; instead, when the panel of 30 sera was tested against the B.1.617.2 (Delta) variant, a significant (p = 0.0015) 2.38-fold reduction in neutralizing titres compared with the reference after the first vaccine dose was demonstrated. After the second vaccine dose, the reduction was not significant (p = 0.1835). This study highlights that the BNT162b2 vaccine stimulates a humoral response able to neutralize all tested SARS-CoV-2 variants, thus suggesting a prominent role in mitigating the impact of the SARS-CoV-2 pandemic in real-world conditions. Long-term follow-up is currently ongoing.     

West Nile Virus Vaccination Protects against Usutu Virus Disease in Mice

West Nile virus (WNV) and Usutu virus (USUV) are mosquito-borne flaviviruses that can cause neuroinvasive disease in humans. WNV and USUV circulate in both Africa and Europe and are closely related. Due to antigenic similarity, WNV-specific antibodies and USUV-specific antibodies have the potential to bind heterologous viruses; however, it is unclear whether this interaction may offer protection against infection. To investigate how prior WNV exposure would influence USUV infection, we used an attenuated WNV vaccine that contains the surface proteins of WNV in the backbone of a dengue virus 2 vaccine strain and protects against WNV disease. We hypothesized that vaccination with this attenuated WNV vaccine would protect against USUV infection. Neutralizing responses against WNV and USUV were measured in vitro using sera following vaccination. Sera from vaccinated CD-1 and Ifnar1^−/− mice cross-neutralized with WNV and USUV. All mice were then subsequently challenged with an African or European USUV strain. In CD-1 mice, there was no difference in USUV titers between vaccinated and mock-vaccinated mice. However, in the Ifnar1^−/− model, vaccinated mice had significantly higher survival rates and significantly lower USUV viremia compared to mock-vaccinated mice. Our results indicate that exposure to an attenuated form of WNV protects against severe USUV disease in mice and elicits a neutralizing response to both WNV and USUV. Future studies will investigate the immune mechanisms responsible for the protection against USUV infection induced by WNV vaccination, providing critical insight that will be essential for USUV and WNV vaccine development.     

Oral immunisation with Taishan Pinus massoniana pollen polysaccharide adjuvant with recombinant Lactococcus lactis-expressing Proteus mirabilis ompA confers optimal protection in mice

BackgroundProteus mirabilis poses a critical burden on the breeding industry, but no efficient vaccine is available for animals.MethodA recombinant Lactococcus lactis expressing the ompA of P. mirabilis was used to develop a vaccine. The mucosal and systemic immune responses of the recombinant vaccine were evaluated in mice after oral immunisation. The inhibition on P. mirabilis colonisation of vaccines was also determined. Moreover, Taishan Pinus massoniana pollen polysaccharides (TPPPS) were used as adjuvants to examine the immunomodulatory effects.ResultsThe pure recombinant L. lactis vaccine significantly induced the production of specific IgA and IgG, IL-2, IL-4, IFN-γ, and T lymphocyte proliferation, and the immunised mice exhibited significant resistance to P. mirabilis colonisation. Notably, the TPPPS adjuvant vaccines induced higher levels of immune responses than the pure L. lactis.ConclusionsThe L. lactis as a vaccine vehicle combined with TPPPS adjuvant provides a feasible method for preventing P. mirabilis infection.     

Immune Responses in Pregnant Sows Induced by Recombinant Lactobacillus johnsonii Expressing the COE Protein of Porcine Epidemic Diarrhea Virus Provide Protection for Piglets against PEDV Infection

Porcine epidemic diarrhea (PED) induced by porcine epidemic diarrhea virus (PEDV) is an intestinal infectious disease in pigs that causes serious economic losses to the pig industry. To develop an effective oral vaccine against PEDV infection, we used a swine-origin Lactobacillus johnsonii (L. johnsonii) as an antigen delivery carrier. A recombinant strain pPG-T7g10-COE/L. johnsonii (L. johnsonii-COE) expressing COE protein (a neutralizing epitope of the viral spike protein) was generated. The immunomodulatory effect on dendritic cell in vitro and immunogenicity in pregnant sows was evaluated following oral administration. L. johnsonii-COE could activate monocyte-derived dendritic cell (MoDC) maturation and triggered cell immune responses. After oral vaccination with L. johnsonii-COE, levels of anti-PEDV-specific serum IgG, IgA, and IgM antibodies as well as mucosal secretory immunoglobulin A (SIgA) antibody were induced in pregnant sows. High levels of PEDV-specific SIgA and IgG antibodies were detected in the maternal milk, which provide effective protection for the piglets against PEDV infection. In summary, oral L. johnsonii-COE was able to efficiently activate anti-PEDV humoral and cellular immune responses, demonstrating potential as a vaccine for use in sows to provide protection of their piglets against PEDV.     

Stromal Interaction Molecule 1 Promotes the Replication of vvIBDV by Mobilizing Ca2+ in the ER

Infectious bursal disease virus (IBDV) is one of the main threats to the poultry industry worldwide. Very virulent IBDV (vvIBDV) is a fatal virus strain that causes heavy mortality in young chicken flocks. Ca²⁺ is one of the most universal and versatile signalling molecules and is involved in almost every aspect of cellular processes. Clinical examination showed that one of the characteristics of vvIBDV-infected chickens was severe metabolic disorders, and the chemical examination showed that their serum Ca²⁺ level decreased significantly. However, there are limited studies on how vvIBDV infection modulates the cellular Ca²⁺ level and the effect of Ca²⁺ level changes on vvIBDV replication. In our study, we found Ca²⁺ levels in the endoplasmic reticulum (ER) of vvIBDV-infected B cells were higher than that of mock-infected cells, and the expression level of stromal interaction molecule 1 (STIM1), an ER Ca²⁺ sensor, was significantly upregulated due to vvIBDV infection. The knock-down expression of STIM1 led to decreased Ca²⁺ level in the ER and suppressed vvIBDV replication, while the over-expressed STIM1 led to ER Ca²⁺ upregulation and promoted vvIBDV replication. We also showed that the inhibition of Ca²⁺-release-activated-Ca²⁺ (CRAC) channels could reduce vvIBDV infection by blocking Ca²⁺ from entering the ER. This study suggests a new mechanism that STIM1 promotes the replication of vvIBDV by mobilizing Ca²⁺ in the ER.     

Genetic characterisation of infectious bursal disease virus isolates in Ethiopia

The objective of the investigation was to characterise infectious bursal disease viruses (IBDV) circulating in commercial and breeding poultry farms in Ethiopia between 2009 and 2011. The nucleotide and deduced amino acid sequence for VP2 hypervariable region of ten IBDVs were determined by RT-PCR, sequenced and compared to well characterised IBDV isolates worldwide. IBDV genetic material was amplified directly from bursa or cell passaged material. Phylogenetically, Ethiopian IBDVs represented two genetic lineages: very virulent (vv) IBDVs or variants of the classical attenuated vaccine strain (D78). The nucleotide identity between Ethiopian vvIBDVs ranged between 0% and 2.6%. Ethiopian vvIBDVs are clustered phylogenetically with the African IBDV genetic lineage, independent of the Asian/European lineage. This report demonstrates the circulation of vvIBDV in commercial and breeding poultry farms in Ethiopia.     

Dynamics of the Emerging Genogroup of Infectious Bursal Disease Virus Infection in Broiler Farms in South Korea: A Nationwide Study

Infectious bursal disease (IBD), caused by IBD virus (IBDV), threatens the health of the poultry industry. Recently, a subtype of genogroup (G) 2 IBDV named G2d has brought a new threat to the poultry industry. To determine the current status of IBDV prevalence in South Korea, active IBDV surveillance on 167 randomly selected broiler farms in South Korea from August 2020 to July 2021 was conducted. The bursas of Fabricius from five chickens from each farm were independently pooled and screened for IBDV using virus-specific RT-PCR. As a result, 86 farms were found to be infected with the G2d variant, 13 farms with G2b, and 2 farms with G3. Current prevalence estimation of IBDV infection in South Korea was determined as 17.8% at the animal level using pooled sampling methods. G2d IBDV was predominant compared to other genogroups, with a potentially high-risk G2d infection area in southwestern South Korea. The impact of IBDV infection on poultry productivity or Escherichia coli infection susceptibility was also confirmed. A comparative pathogenicity test indicated that G2d IBDV caused severe and persistent damage to infected chickens compared with G2b. This study highlights the importance of implementation of regular surveillance programs and poses challenges for the comprehensive prevention of IBDV infections.     

Differential Modulation of Innate Antiviral Profiles in the Intestinal Lamina Propria Cells of Chickens Infected with Infectious Bursal Disease Viruses of Different Virulence

Infectious bursal disease virus (IBDV) is one of the most important infectious diseases of poultry around the world. Gut-associated lymphoid tissues (GALT) are the first line of defense of the host against the infection. The purpose of this study was to investigate the role of innate immune antiviral signaling triggered by Toll-like receptor 3 (TLR3), as well as macrophage activation and cytokine response in the intestinal lamina propria (ILP) cells after the oral challenge of IBDV in relation to IBDV virulence and disease pathogenesis. The results showed that the expression levels of TLR3, IRF7, IFN-α/β and the corresponding downstream antiviral factors OAS, PKR and Mx were all upregulated in the SPF chicken ILP cells at 8 h post-infection (hpi) and 12 hpi. Similarly, macrophages were activated, with the initial macrophage M1 activation observed at 8 hpi, but then it rapidly shifted to a non-protective M2-type. Both Th1 (IFN-γ, TNF-α, IL-12) and Th2 (IL-4 and IL-10) types of cytokines were differentially upregulated during the early stage of infection; however, the Th1 cytokines exhibited stronger activation before 8 hpi compared to those of the Th2 cytokines. Interestingly, differential regulations of gene expression induced by different IBDV strains with different virulence were detected. The HLJ0504-like very virulent (vv) IBDV strain NN1172 induced stronger activation of TLR3-IFN-α/β pathway, macrophages and the Th1/2 cytokines’ expression, compared to those induced by the attenuated strain B87 at 8 hpi and 12 hpi in the ILP cells. In conclusion, the innate antiviral response mediated by the TLR3-IRF7 pathway, macrophage activation and cytokine expression in the GALT cells at the early stage of IBDV infection was differentially modulated, and the HLJ0504-like vvIBDV strain triggered stronger activation than the attenuated vaccine strain, and that may play an important role in the progression of disease.     

Clusters of SARS-CoV-2 Lineage B.1.1.7 Infection after Vaccination with Adenovirus-Vectored and Inactivated Vaccines

A SARS-CoV-2 B.1.1.7 variant of concern (VOC) has been associated with increased transmissibility, hospitalization, and mortality. This study aimed to explore the factors associated with B.1.1.7 VOC infection in the context of vaccination. On March 2021, we detected SARS-CoV-2 RNA in nasopharyngeal samples from 14 of 22 individuals vaccinated with a single-dose of ChAdOx1 (outbreak A, n = 26), and 22 of 42 of individuals with two doses of the CoronaVac vaccine (outbreak B, n = 52) for breakthrough infection rates for ChAdOx1 of 63.6% and 52.4% for CoronaVac. The outbreaks were caused by two independent clusters of the B.1.1.7 VOC. The serum of PCR-positive symptomatic SARS-CoV-2-infected individuals had ~1.8–3.4-fold more neutralizing capacity against B.1.1.7 compared to the serum of asymptomatic individuals. These data based on exploratory analysis suggest that the B.1.1.7 variant can infect individuals partially immunized with a single dose of an adenovirus-vectored vaccine or fully immunized with two doses of an inactivated vaccine, although the vaccines were able to reduce the risk of severe disease and death caused by this VOC, even in the elderly.     

SARS-CoV-2 Gamma and Delta Variants of Concern Might Undermine Neutralizing Activity Generated in Response to BNT162b2 mRNA Vaccination

The Delta variant raised concern regarding its ability to evade SARS-CoV-2 vaccines. We evaluated a serum neutralizing response of 172 Italian healthcare workers, three months after complete Comirnaty (BNT162b2 mRNA, BioNTech-Pfizer) vaccination, testing their sera against viral isolates of Alpha, Gamma and Delta variants, including 36 subjects with a previous SARS-CoV-2 infection. We assessed whether IgG anti-spike TRIM levels and serum neutralizing activity by seroneutralization assay were associated. Concerning Gamma variant, a two-fold reduction in neutralizing titres compared to the Alpha variant was observed, while a four-fold reduction of Delta virus compared to Alpha was found. A gender difference was observed in neutralizing titres only for the Gamma variant. The serum samples of 36 previously infected SARS-CoV-2 individuals neutralized Alpha, Gamma and Delta variants, demonstrating respectively a nearly three-fold and a five-fold reduction in neutralizing titres compared to Alpha variant. IgG anti-spike TRIM levels were positively correlated with serum neutralizing titres against the three variants. The Comirnaty vaccine provides sustained neutralizing antibody activity towards the Alpha variant, but it is less effective against Gamma and even less against Delta variants.     

The E484K Substitution in a SARS-CoV-2 Spike Protein Subunit Vaccine Resulted in Limited Cross-Reactive Neutralizing Antibody Responses in Mice

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), especially emerging variants, poses an increased threat to global public health. The significant reduction in neutralization activity against the variants such as B.1.351 in the serum of convalescent patients and vaccinated people calls for the design of new potent vaccines targeting the emerging variant. However, since most vaccines approved and in clinical trials are based on the sequence of the original SARS-CoV-2 strain, the immunogenicity and protective efficacy of vaccines based on the B.1.351 variant remain largely unknown. In this study, we evaluated the immunogenicity, induced neutralization activity, and protective efficacy of wild-type spike protein nanoparticle (S-2P) and mutant spike protein nanoparticle (S-4M-2P) carrying characteristic mutations of B.1.351 variant in mice. Although there was no significant difference in the induction of spike-specific IgG responses in S-2P- and S-4M-2P-immunized mice, neutralizing antibodies elicited by S-4M-2P exhibited noteworthy, narrower breadth of reactivity with SARS-CoV-2 variants compared with neutralizing antibodies elicited by S-2P. Furthermore, the decrease of induced neutralizing antibody breadth at least partly resulted from the amino acid substitution at position 484. Moreover, S-4M-2P vaccination conferred insufficient protection against live SARS-CoV-2 virus infection, while S-2P vaccination gave definite protection against SARS-CoV-2 challenge in mice. Together, our study provides direct evidence that the E484K substitution in a SARS-CoV-2 subunit protein vaccine limited the cross-reactive neutralizing antibody breadth in mice and, more importantly, draws attention to the unfavorable impact of this mutation in spike protein of SARS-CoV-2 variants on the induction of potent neutralizing antibody responses.     

Structural basis for differential neutralization of ebolaviruses

There are five antigenically distinct ebolaviruses that cause hemorrhagic fever in humans or non-human primates (Ebola virus, Sudan virus, Reston virus, Taï Forest virus, and Bundibugyo virus). The small handful of antibodies known to neutralize the ebolaviruses bind to the surface glycoprotein termed GP_1,2. Curiously, some antibodies against them are known to neutralize in vitro but not protect in vivo, whereas other antibodies are known to protect animal models in vivo, but not neutralize in vitro. A detailed understanding of what constitutes a neutralizing and/or protective antibody response is critical for development of novel therapeutic strategies. Here, we show that paradoxically, a lower affinity antibody with restricted access to its epitope confers better neutralization than a higher affinity antibody against a similar epitope, suggesting that either subtle differences in epitope, or different characteristics of the GP_1,2 molecules themselves, confer differential neutralization susceptibility. Here, we also report the crystal structure of trimeric, prefusion GP_1,2 from the original 1976 Boniface variant of Sudan virus complexed with 16F6, the first antibody known to neutralize Sudan virus, and compare the structure to that of Sudan virus, variant Gulu. We discuss new structural details of the GP₁-GP₂ clamp, thermal motion of various regions in GP_1,2 across the two viruses visualized, details of differential interaction of the crystallized neutralizing antibodies, and their relevance for virus neutralization.     

Glycosite-deleted mRNA of SARS-CoV-2 spike protein as a broad-spectrum vaccine

Development of the messenger RNA (mRNA) vaccine has emerged as an effective and speedy strategy to control the spread of new pathogens. After vaccination, the mRNA is translated into the real protein vaccine, and there is no need to manufacture the protein in vitro. However, the fate of mRNA and its posttranslational modification inside the cell may affect immune response. Here, we showed that the mRNA vaccine of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike protein with deletion of glycosites in the receptor-binding domain (RBD) or especially the subunit 2 (S2) domain to expose more conserved epitopes elicited stronger antibody and CD8⁺ T cell responses with broader protection against the alpha, beta, gamma, delta, and omicron variants, compared to the unmodified mRNA. Immunization of such mRNA resulted in accumulation of misfolded spike protein in the endoplasmic reticulum, causing the up-regulation of BiP/GRP78, XBP1, and p-eIF2α to induce cell apoptosis and strong CD8⁺ T cell response. In addition, dendritic cells (DCs) incubated with S2-glysosite deleted mRNA vaccine increased class I major histocompatibility complex (MHC I) expression. This study provides a direction for the development of broad-spectrum mRNA vaccines which may not be achieved with the use of expressed proteins as antigens.

In 1796, Edward Jenner created the first vaccine (cowpox) in the world to protect against smallpox and successfully rescued millions of people, and, since then, vaccination has been recognized as the best way to protect against pathogens (1). Since the outbreak of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in December 2019 that caused COVID-19, the virus has spread all over the world and caused more than 200 million infections and 4 million deaths in 20 mo (2–4). Of the various vaccines developed to control the spread of SARS-CoV-2 and variants, the messenger RNA (mRNA) vaccines developed by Moderna and BioNTech/Pfizer have proven highly effective and represent a major breakthrough in vaccine development, mainly due to the speed and convenience. These vaccines were stabilized with new mRNA technology and lipid nanoparticle (LNP) formulation for delivery and translation into the spike (S) protein in vivo to induce immune response (5–7). The nucleoside modification, sequence optimization, synthesis, and high-performance liquid chromatography purification were the major steps used for development of mRNA vaccines. However, after mRNA vaccination, the translational process and posttranslational modification of the expressed immunogen, and how they affect the outcome of immune response, are not well understood (8). Among the many posttranslational modification events, glycosylation is known to play an important role in protein folding, structure, and function (9). Our previous study showed that deletion of certain glycosites in the S protein affected the expression and folding of S protein, making it difficult to prepare pure S proteins with deletion of specific glycosylation sites for the study of their receptor binding and immune response (10). Therefore, in this study, the mRNA of SARS-CoV-2 S protein with deletion of specific glycosites is used to investigate the effect of glycosylation on protein expression and immune response in vivo.The SARS-CoV-2 S protein has three major immunogenic domains: the N-terminal domain, the receptor-binding domain (RBD), and the subunit 2 domain (S2) (11). Previous studies have shown that the neutralizing antibodies that recognize the RBD are highly protective against SARS-CoV-2 and other coronaviruses (12–14). However, the efficacy of antibodies against the emerging variants was found to decrease as the virus continued to mutate. More than 3 million S protein sequences have been reported to the global database Global Initiative on Sharing Avian Influenza Database (GISAID), and all are highly glycosylated, with 22 N-glycosites and 2 O-glycosites per monomer. As part of our efforts to develop broadly protective vaccines and antibodies against SARS-CoV-2 and the emerging variants, we have shown that immunization of wild-type (WT) S protein with all 22 N-glycosites trimmed down to N-acetylglucosamine (GlcNAc) as the mono-GlcNAc decorated S protein (S_mg) elicited broadly protective immune responses, including antibody and CD4⁺ as well as CD8⁺ T cell responses against the alpha, beta, gamma, and delta variants. Further study showed that most of the conserved epitopes on S protein are located in the RBD and the HR2 domain of the S2 subunit, but these conserved epitopes are largely shielded by glycans to escape from the immune response (10). So, removal of the shielded glycans to expose more conserved epitopes induced broader and stronger immune responses. We also used the single B cell technology to screen B cell clones from S_mg immunized mice, and we identified a broadly neutralizing monoclonal antibody targeting a highly conserved epitope in RBD which was not found in S_fg immunized mice, further demonstrating that removal of glycan shields from S protein is an effective strategy for development of a broadly protective vaccine against SARS-CoV-2 variants (10). To translate this finding into the mRNA vaccine design for development of a broad-spectrum mRNA vaccine, here we show the study of S protein mRNA vaccine with deletion of specific glycosites in RBD and S2 with N to Q and S/T to A replacement and investigation of their protein expression and immune response as well as breadth of protection.     

Cellular immune responses to human papillomavirus (HPV)-16 L1 in healthy volunteers immunized with recombinant HPV-16 L1 virus-like particles

The causal association between papillomavirus (HPV) infection and cervical cancer has been demonstrated; the development of a prophylactic vaccine to protect against HPV infection may therefore reduce the incidence of this cancer worldwide. Noninfectious HPV-like particles (VLPs), composed of the L1 major capsid protein, are current candidate vaccines for prevention of HPV infection and cervical neoplasia. Although neutralizing antibodies have a pivotal role in the prevention of initial infection, cellular immune responses to HPV antigens may have an important role in viral clearance. A phase II trial was conducted to further evaluate the immunogenicity of a recombinant HPV-16 L1 VLP vaccine administered intramuscularly, without adjuvant, at 0, 1, and 6 months. Cell-mediated immune responses (lymphoproliferation and cytokine production) to HPV-16 L1 VLPs were evaluated in peripheral blood mononuclear cells (PBMCs) from 43 individuals receiving the L1 VLP vaccine and from 10 individuals receiving placebo. Vaccination resulted, at months 2 and 7 (i.e., 1 month after the second immunization and 1 month after third immunization, respectively), in increases in T cell-proliferative response to HPV-16 L1 VLPs (P<.001). In addition, significant increases in cytokine (interferon-gamma, interleukin [IL]-5 and IL-10) responses to L1 VLPs were observed after vaccination (P<.001). The strongest cytokine responses at month 7 were observed in individuals with high antibody titers at month 2, suggesting that neutralizing antibodies generated by initial vaccination may augment T cell responses to subsequent booster vaccinations. No significant increases in lymphoproliferative or cytokine responses to L1 VLPs were observed in individuals receiving placebo. In summary, the HPV-16 L1 vaccine induces not only robust B cell responses but also L1-specific T cell responses detectable by proliferation of both CD4+ and CD8+ T cells and in vitro production of both Th1- and Th2-type cytokines. Future efficacy studies are needed to evaluate whether and/or how VLP vaccines confer protection against genital HPV infection and associated disease.     

Perspectives for the utilization of neutralizing human monoclonal antibodies as anti-HCV drugs

Broadly neutralizing antibodies protect against hepatitis C virus quasispecies challenge. Law M, Maruyama T, Lewis J, Giang E, Tarr AW, Stamataki Z, Gastaminza P, Chisari FV, Jones IM, Fox RI, Ball JK, McKeating JA, Kneteman NM, Burton DR. A major problem in hepatitis C virus (HCV) immunotherapy or vaccine design is the extreme variability of the virus. We identified human monoclonal antibodies (mAbs) that neutralize genetically diverse HCV isolates and protect against heterologous HCV quasispecies challenge in a human liver-chimeric mouse model. The results provide evidence that broadly neutralizing antibodies to HCV protect against heterologous viral infection and suggest that a prophylactic vaccine against HCV may be achievable. [Abstract reproduced by permission of Nat Med 2008;14:25-27].     

Seroprevalence of SARS-CoV-2 IgG Antibodies and Factors Associated with SARS-CoV-2 IgG Neutralizing Activity among Primary Health Care Workers 6 Months after Vaccination Rollout in France

We aimed to investigate the immunoglobulin G response and neutralizing activity against severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) among primary health care workers (PHCW) in France and assess the association between the neutralizing activity and several factors, including the coronavirus disease 2019 (COVID-19) vaccination scheme. A cross-sectional survey was conducted between 10 May 2021 and 31 August 2021. Participants underwent capillary blood sampling and completed a questionnaire. Sera were tested for the presence of antibodies against the nucleocapsid (N) protein and the S-1 portion of the spike (S) protein and neutralizing antibodies. In total, 1612 PHCW were included. The overall seroprevalences were: 23.6% (95% confidence interval (CI) 21.6–25.7%) for antibodies against the N protein, 94.7% (93.6–95.7%) for antibodies against the S protein, and 81.3% (79.4–83.2%) for neutralizing antibodies. Multivariate regression analyses showed that detection of neutralizing antibodies was significantly more likely in PHCW with previous SARS-CoV-2 infection than in those with no such history among the unvaccinated (odds ratio (OR) 16.57, 95% CI 5.96–59.36) and those vaccinated with one vaccine dose (OR 41.66, 95% CI 16.05–120.78). Among PHCW vaccinated with two vaccine doses, the detection of neutralizing antibodies was not significantly associated with previous SARS-CoV-2 infection (OR 1.31, 95% CI 0.86–2.07), but was more likely in those that received their second vaccine dose within the three months before study entry than in those vaccinated more than three months earlier (OR 5.28, 95% CI 3.51–8.23). This study highlights that previous SARS-CoV-2 infection and the time since vaccination should be considered when planning booster doses and the design of COVID-19 vaccine strategies.     

Phlebotomus papatasi exposure cross-protects mice against Leishmania major co-inoculated with Phlebotomus duboscqi salivary gland homogenate

Leishmania parasites are inoculated into host skin together with sand fly saliva and multiple exposures to uninfected sand fly bites protect mice against Leishmania infection. However, sand fly vectors differ in composition of the saliva and therefore the protection elicited by their salivary proteins was shown to be species-specific. On the other hand, the optimal vaccine based on sand fly salivary proteins should be based on conserved salivary proteins conferring cross-reactivity. In the present study we therefore focused on cross-protective properties of saliva from Phlebotomus papatasi and Phlebotomus duboscqi, the two natural vectors of Leishmania major. Two groups of mice exposed to bites of P. papatasi and two control, non-immunized groups were infected with L. major promastigotes along with either P. papatasi or P. duboscqi salivary gland homogenate. All mice were followed for the development of Leishmania lesions, parasite burdens, specific antibodies, and for production of NO, urea, or cytokines by peritoneal macrophages. Protection against Leishmania infection was observed not only in exposed mice challenged with homologous saliva but also in the group challenged with P. duboscqi saliva. Comparing both exposed groups, no significant differences were observed in parasite load, macrophage activity, or in the levels of anti-L. major and anti-P. papatasi/P. duboscqi antibodies. This is the first study showing cross-protection caused by salivary antigens of two Phlebotomus species. The cross-protective effect suggests that the anti-Leishmania vaccine based on P. papatasi salivary proteins might be applicable also in areas where L. major is transmitted by P. duboscqi.     

Characterization of Near Full-Length Transmitted/Founder HIV-1 Subtype D and A/D Recombinant Genomes in a Heterosexual Ugandan Population (2006–2011)

Detailed characterization of transmitted HIV-1 variants in Uganda is fundamentally important to inform vaccine design, yet studies on the transmitted full-length strains of subtype D viruses are limited. Here, we amplified single genomes and characterized viruses, some of which were previously classified as subtype D by sub-genomic pol sequencing that were transmitted in Uganda between December 2006 to June 2011. Analysis of 5′ and 3′ half genome sequences showed 73% (19/26) of infections involved single virus transmissions, whereas 27% (7/26) of infections involved multiple variant transmissions based on predictions of a model of random virus evolution. Subtype analysis of inferred transmitted/founder viruses showed a high transmission rate of inter-subtype recombinants (69%, 20/29) involving mainly A1/D, while pure subtype D variants accounted for one-third of infections (31%, 9/29). Recombination patterns included a predominance of subtype D in the gag/pol region and a highly recombinogenic envelope gene. The signal peptide-C1 region and gp41 transmembrane domain (Tat2/Rev2 flanking region) were hotspots for A1/D recombination events. Analysis of a panel of 14 transmitted/founder molecular clones showed no difference in replication capacity between subtype D viruses (n = 3) and inter-subtype mosaic recombinants (n = 11). However, individuals infected with high replication capacity viruses had a faster CD4 T cell loss. The high transmission rate of unique inter-subtype recombinants is striking and emphasizes the extraordinary challenge for vaccine design and, in particular, for the highly variable and recombinogenic envelope gene, which is targeted by rational designs aimed to elicit broadly neutralizing antibodies.