Comparative Evaluation of Two Vaccine Candidates against Experimental Leishmaniasis Due to Leishmania major Infection in Four Inbred Mouse Strains

Experimental leishmaniasis in BALB/c and C57BL/6 mice are the most investigated murine models that were used for the preclinical evaluation of Leishmania vaccine candidates. We have previously described two new inbred mouse strains named PWK and MAI issued from feral founders that also support the development of experimental leishmaniasis due to L. major. In this study, we sought to determine whether different mouse inbred strains generate concordant or discordant results when used to evaluate the potential of Leishmania proteins to protect against experimental leishmaniasis. To this end, two Leishmania proteins, namely, LACK (for Leishmania homolog of receptor for activated C kinase) and LmPDI (for L. major protein disulfide isomerase) were compared for their capacity to protect against experimental leishmaniasis in PWK, MAI, BALB/c, and C57BL/6 inbred mouse strains. Our data show that the capacity of Leishmania proteins to confer protection depends on the mouse strain used, stressing the important role played by the genetic background in shaping the immune response against the pathogen. These results may have important implications for the preclinical evaluation of candidate Leishmania vaccines: rather than using a single mouse strain, a panel of different inbred strains of various genetic backgrounds should be tested in parallel. The antigen that confers protection in the larger range of inbred strains may have better chances to be also protective in outbred human populations and should be selected for clinical trials.The leishmaniasis are parasitic diseases due to a protozoan of the genus Leishmania that are endemic in 88 countries. Three hundred and fifty million people are exposed to the infection risk and 14 million people are known to be infected. Two million new cases, including 1.5 million of the cutaneous leishmaniasis, are estimated to appear annually (39). The leishmaniasis represent a worldwide major public health problem because of several therapeutic challenges such as drug toxicity, parasite resistance to current drugs, and the high cost of the new treatments. The problem is particularly serious since the disease affects the poorest classes of the developing countries. The cutaneous leishmaniasis are among the rare parasitic diseases that might be potentially vaccine preventable. However, even if theoretically feasible, there is still no human Leishmania vaccine available today (17). One serious obstacle facing such a goal is the lack of experimental animal models that tightly mimic the disease as it occurs in humans.The experimental infection of inbred BALB/c and C57BL/6 mice by Leishmania major parasites has established the functionality of the Th1/Th2 dichotomy of CD4⁺ T helper cells and the contrasted pathogenic roles played in protection or disease promotion by the two Th subsets (33). Thus, C57BL/6 mice infected with L. major develop a Th1 response and efficiently control the disease within few weeks. In contrast, susceptible BALB/c mice mount a Th2 response and develop a severe, unremitting, and ultimately lethal disease (37). The susceptibility of BALB/c mice to L. major infection has been ascribed to the occurrence within the lymph nodes draining the inoculation site, of an early burst (at 16 h postinoculation) of interleukin-4 (IL-4) that polarizes the immune response toward the Th2 pathway (15, 24). The contrasted immunopathogenic mechanisms at work in BALB/c and C57BL/6 strains likely reflect differences in their genetic background. Since the majority of studies evaluating vaccine candidates have been conducted in the BALB/c model, it would be hazardous to extrapolate the conclusions drawn from these experiments to other inbred strains of different genetic backgrounds or to out bred animal models (i.e., primates): one given vaccine could be promising in one strain and still fail to protect in another strain (17). Thus, the criteria that would help to select at the preclinical stage a Leishmania antigen as a promising vaccine candidate worth entering the clinical trial stage are still not clearly defined.We have recently identified two new inbred mouse strains derived from feral founders, named PWK and MAI, that are susceptible to L. major infection (1). MAI mice develop an infiltrated lesion at the site of parasite inoculation that enlarges over time in an unremitted way. In this strain, the primary infection does not induce protection against reinfection. Although the immune response to Leishmania antigens in MAI mice was characterized by a Th2 cytokine profile, IL-4 did not seem to play a dominant role in disease phenotype as in BALB/c mice. In PWK mice, the experimental disease induced by L. major infection is featured by a nodule that develops at the site of parasite inoculation. This nodule is larger and of a much longer duration (30 weeks to complete healing) than the one that develops in C57BL/6. PWK mice acquire a solid immunity after a primary infection and are completely refractory to a secondary challenge. They develop during infection a mixed Th1/Th2 cytokine pattern, with IL-10 playing a disease-promoting role.The diverse disease patterns induced by L. major in PWK, MAI, C57BL/6, and BALB/c mice and the heterogeneity in the immunopathogenic mechanisms at work in each strain are likely shaped by the genetic background of the mice. This assumption led us to explore the effect of the genetic diversity of inbred mouse strains on the protection potentially conferred by Leishmania proteins against L major infection. Two Leishmania promising vaccine candidates were used, namely, the Leishmania homolog of receptor for activated C kinase (LACK) (31) and the L. major protein disulfide isomerase (LmPDI) (5).     

Safety and Immunogenicity of Two RNA-Based Covid-19 Vaccine Candidates

Background:Severe acute respiratory syndrome coronavirus 2(SARS-CoV-2)infections and the resulting disease,coronavirus disease 2019(Covid-19),have spread to millions of persons worldwide.Multiple vaccine candidates are under development,but no vaccine is currently available.Interim safety and immunogenicity data about the vaccine candidate BNT162b1 in younger adults have been reported previously from trials in Germany and the United States.     

Transplacental Transmission of Venezuelan Equine Encephalomyelitis Virus in Mice

Transplacental infection of mouse fetuses with Venezuelan equine encephalomyelitis was produced by intraperitoneal injection of dams at various stages of gestation with 10(3) suckling mouse intracerebral median lethal doses of an attenuated strain (TC-83). Virus inoculation, at times ranging from 6 days prior to mating to 9 days after conception, had no effect on conception rate, litter size, or survival of the newborn. Inoculation of the dam from the 10th to 13th days of gestation resulted in decreased litter size, an increase in stillbirths, and a decrease in birth-to-weaning survival. Inoculation of the dams later in gestation only decreased the birth-to-weaning survival. No evidence of morphologic abnormality was noted in any of the newborn.     

Evaluation of the Proline-Rich Antigen of Coccidioides immitis as a Vaccine Candidate in Mice

We have expressed the proline-rich antigen (PRA) from Coccidioides immitis in Escherichia coli and evaluated its potential as a vaccine candidate. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of the recombinant protein (rPRA) revealed two bands, which exhibited virtually identical primary amino acid sequences. T cells from rPRA-immunized BALB/c mice showed a significant in vitro proliferative response to rPRA. A small but statistically significant proliferative response was also induced by rPRA in T cells from mice immunized with whole-cell coccidioidal vaccines. BALB/c mice immunized with rPRA and challenged intraperitoneally with virulent C. immitis had a greatly reduced fungal burden in their lungs and spleens compared to unvaccinated mice. The number of organisms in the lungs was reduced 500-fold, and similar reductions were observed in the spleens of immunized mice. These studies support the continued development of rPRA as a candidate vaccine for prevention of coccidioidomycosis.     

Comparative Efficacy of Two Leading Candidate Ricin Toxin A Subunit Vaccines in Mice

The two leading ricin toxin vaccine candidates, RVEc and RiVax, are recombinant derivatives of the toxin''s 267-amino-acid enzymatic A chain (RTA). RVEc is truncated at the C terminus (residues 199 to 267) to improve protein thermostability, while RiVax has two point mutations (V76M and Y80A) that eliminate the RNA N-glycosidase activity of RTA, as well as its ability to induce vascular leak syndrome. The two vaccines have never been directly compared in terms of their ability to stimulate RTA-specific antibodies (Abs), toxin-neutralizing activity (TNA), or protective immunity. To address this issue, groups of female BALB/c mice were immunized two or three times with Alhydrogel-adsorbed RiVax or RVEc at a range of doses (0.3 to 20 μg) and then challenged with 10 50% lethal doses (LD₅₀s) of ricin. We found that the vaccines were equally effective at eliciting protective immunity at the doses tested. There were, however, quantitative differences in the antibody responses. RVEc tended to elicit higher levels of ricin-specific RTA IgG and TNA than did RiVax. Pepscan analysis revealed that serum Abs elicited by RVEc were skewed toward a solvent-exposed immunodominant α-helix known to be the target of potent toxin-neutralizing Abs. Finally, immunodepletion experiments suggest that the majority of toxin-neutralizing Abs elicited by RiVax were confined to residues 1 to 198, possibly explaining the equal effectiveness of RVEc as a vaccine.     

Comparative Genomics and Immunoinformatics Approach for the Identification of Vaccine Candidates for Enterohemorrhagic Escherichia coli O157:H7

Enterohemorrhagic Escherichia coli (EHEC) O157:H7 strains are major human food-borne pathogens, responsible for bloody diarrhea and hemolytic-uremic syndrome worldwide. Thus far, there is no vaccine for humans against EHEC infections. In this study, a comparative genomics analysis was performed to identify EHEC-specific antigens useful as potential vaccines. The genes present in both EHEC EDL933 and Sakai strains but absent in nonpathogenic E. coli K-12 and HS strains were subjected to an in silico analysis to identify secreted or surface-expressed proteins. We obtained a total of 65 gene-encoding protein candidates, which were subjected to immunoinformatics analysis. Our criteria of selection aided in categorizing the candidates as high, medium, and low priority. Three members of each group were randomly selected and cloned into pVAX-1. Candidates were pooled accordingly to their priority group and tested for immunogenicity against EHEC O157:H7 using a murine model of gastrointestinal infection. The high-priority (HP) pool, containing genes encoding a Lom-like protein (pVAX-31), a putative pilin subunit (pVAX-12), and a fragment of the type III secretion structural protein EscC (pVAX-56.2), was able to induce the production of EHEC IgG and sIgA in sera and feces. HP candidate-immunized mice displayed elevated levels of Th2 cytokines and diminished cecum colonization after wild-type challenge. Individually tested HP vaccine candidates showed that pVAX-12 and pVAX-56.2 significantly induced Th2 cytokines and production of fecal EHEC sIgA, with pVAX-56.2 reducing EHEC cecum colonization. We describe here a bioinformatics approach able to identify novel vaccine candidates potentially useful for preventing EHEC O157:H7 infections.     

灭活SARS病毒的免疫原性的实验研究

研究灭活SARS病毒的免疫原性。SARS病毒F6 9株经甲醛灭活后 ,加入氢氧化铝佐剂 ,以 3种剂量接种BALB/c小鼠 ,定时采血 ,测定特异性抗体的滴度及其中和活性 ,同时用化学发光酶联免疫法测定抗血清与SARS病毒结构蛋白的反应特异性及其相对强度。结果发现 ,小鼠接种疫苗 4d后 ,血清中可检测到IgM抗体 ,直至 2 6d后逐渐下降 ;IgG抗体在初次免疫 8d后出现 ,4 7d时达最高峰 ,6 3d后进入稳定期 ;不同剂量组的抗体滴度具有明显剂效关系 ,低剂量组和中剂量组滴度峰值为 1∶192 0 0 ,高剂量组滴度峰值为 1∶384 0 0。中和实验结果表明 ,小鼠所产生的抗体具有中和病毒活性 ,在 6 3d时 ,低剂量组和中剂量组血清的中和效价为 1∶12 80 ,高剂量组血清的中和效价为 1∶5 12 0。抗体分类结果表明 ,小鼠抗血清中含有针对多种SARS病毒结构蛋白的特异性抗体 ,其中 ,针对N蛋白、S4蛋白和S2蛋白的抗体水平相对较高 ,而抗M抗体、抗 3CL抗体的水平相对较低。上述结果说明 ,SARS病毒F6 9株经甲醛灭活后 ,各主要结构蛋白仍保持较强免疫原性 ;免疫小鼠后 ,可以诱导产生高滴度的特异性混合抗体 ,在体外可以保护敏感细胞不受SARS病毒攻击     

Evaluation of Protective Potential of Yersinia pestis Outer Membrane Protein Antigens as Possible Candidates for a New-Generation Recombinant Plague Vaccine

Plague caused by Yersinia pestis manifests itself in bubonic, septicemic, and pneumonic forms. Although the U.S. Food and Drug Administration recently approved levofloxacin, there is no approved human vaccine against plague. The capsular antigen F1 and the low-calcium-response V antigen (LcrV) of Y. pestis represent excellent vaccine candidates; however, the inability of the immune responses to F1 and LcrV to provide protection against Y. pestis F1⁻ strains or those which harbor variants of LcrV is a significant concern. Here, we show that the passive transfer of hyperimmune sera from rats infected with the plague bacterium and rescued by levofloxacin protected naive animals against pneumonic plague. Furthermore, 10 to 12 protein bands from wild-type (WT) Y. pestis CO92 reacted with the aforementioned hyperimmune sera upon Western blot analysis. Based on mass spectrometric analysis, four of these proteins were identified as attachment invasion locus (Ail/OmpX), plasminogen-activating protease (Pla), outer membrane protein A (OmpA), and F1. The genes encoding these proteins were cloned, and the recombinant proteins purified from Escherichia coli for immunization purposes before challenging mice and rats with either the F1⁻ mutant or WT CO92 in bubonic and pneumonic plague models. Although antibodies to Ail and OmpA protected mice against bubonic plague when challenged with the F1⁻ CO92 strain, Pla antibodies were protective against pneumonic plague. In the rat model, antibodies to Ail provided protection only against pneumonic plague after WT CO92 challenge. Together, the addition of Y. pestis outer membrane proteins to a new-generation recombinant vaccine could provide protection against a wide variety of Y. pestis strains.     

Enhancement, by Two Carboxylic Acid Interferon Inducers, of Resistance Stimulated in Mice by Foot-and-Mouth Disease Vaccine

Simultaneous injection of divinyl ether-maleic amhydride (DVE/MA) or itaconic-acrylic acid and foot-and-mouth disease virus vaccine enhanced the survival of infant mice to subsequent injections of virus. This enhanced resistance was obtained even with doses of interferon inducers which, when administered alone, failed to protect the mice. There was some increase in serum-neutralizing antibody in mice 7 days after injection of DVE/MA and vaccine, as compared with mice given vaccine alone, but there was no clear connection between antibody level and amount of DVE/MA administered.     

Live Oral Cholera Vaccine: Evaluation of the Clinical Effectiveness of Two Strains in Humans

El Tor Ogawa C14-S5 and EW-6, two live vaccine candidate strains, were given to volunteers in varying doses with and without bicarbonate. Vibrios were found in the stool of one of 32 men given the vaccine strain, and only three men developed a significant titer rise (fourfold or greater) at 2 weeks of vibriocidal or antitoxic antibody. Five men who had previously received 10⁹ organisms of the C14-S5 strain were challenged subsequently with virulent Ogawa 395 Vibrio cholerae. The rate of clinical infection in these men was no different than in unvaccinated controls. It was demonstrated that the live oral cholera vaccines did not remain viable in the intestine long enough to act antigenically.     

Characterization of Novel Multiantigenic Vaccine Candidates with Pan-HLA Coverage against Mycobacterium tuberculosis

The low protection by the bacillus Calmette-Guérin (BCG) vaccine and existence of drug-resistant strains require better anti-Mycobacterium tuberculosis vaccines with a broad, long-lasting, antigen-specific response. Using bioinformatics tools, we identified five 19- to 40-mer signal peptide (SP) domain vaccine candidates (VCs) derived from M. tuberculosis antigens. All VCs were predicted to have promiscuous binding to major histocompatibility complex (MHC) class I and II alleles in large geographic territories worldwide. Peripheral mononuclear cells (PBMC) from healthy naïve donors and tuberculosis patients exhibited strong proliferation that correlated positively with Th1 cytokine secretion only in healthy naïve donors. Proliferation to SP VCs was superior to that to antigen-matched control peptides with similar length and various MHC class I and II binding properties. T-cell lines induced to SP VCs from healthy naïve donors had increased CD44^high/CD62L⁺ activation/effector memory markers and gamma interferon (IFN-γ), but not interleukin-4 (IL-4), production in both CD4⁺ and CD8⁺ T-cell subpopulations. T-cell lines from healthy naïve donors and tuberculosis patients also manifested strong, dose-dependent, antigen-specific cytotoxicity against autologous VC-loaded or M. tuberculosis-infected macrophages. Lysis of M. tuberculosis-infected targets was accompanied by high IFN-γ secretion. Various combinations of these five VCs manifested synergic proliferation of PBMC from selected healthy naïve donors. Immunogenicity of the best three combinations, termed Mix1, Mix2, and Mix3 and consisting of 2 to 5 of the VCs, was then evaluated in mice. Each mixture manifested strong cytotoxicity against M. tuberculosis-infected macrophages, while Mix3 also manifested a VC-specific humoral immune response. Based on these results, we plan to evaluate the protection properties of these combinations as an improved tuberculosis subunit vaccine.     

Evaluation of Clumping Factor A Binding Region A in a Subunit Vaccine against Staphylococcus aureus-Induced Mastitis in Mice

The present study evaluated the potential of recombinant binding region A of clumping factor A (rClfA-A) to be an effective component of a vaccine against mastitis induced by Staphylococcus aureus in the mouse. rClfA-A and inactivated S. aureus were each emulsified in Freund''s adjuvant, mineral oil adjuvant, and Seppic adjuvant; phosphate-buffered saline was used as a control. Seven groups of 12 mice each were immunized intraperitoneally three times at 2-week intervals. The titers of IgG and subtypes thereof (IgG1 and IgG2a) in the rClfA-A-immunized group were more than 1,000-fold higher than those in the killed-bacteria-immunized group (P < 0.01). Of the three adjuvants used, mineral oil adjuvant induced the highest antibody levels for both antigens (P < 0.001). Furthermore, the anti-rClfA-A antibody capacities for bacterial adhesion and opsonizing phagocytosis were significantly greater in the rClfA-A-immunized group than in the killed-bacteria-immunized group (P < 0.05). Lactating mice immunized with either rClfA-A or inactivated vaccine were challenged with S. aureus via the intramammary route. The numbers of bacteria recovered from the murine mammary glands 24 h after inoculation were significantly lower in the rClfA-A group than in the killed-bacteria-immunized group (P < 0.001). Histologic examination of the mammary glands showed that rClfA-A immunization effectively preserved tissue integrity. Thus, rClfA-A emulsified in an oil adjuvant provides strong immune protection against S. aureus-induced mastitis in the mouse.Staphylococcus aureus is the most common etiologic agent of contagious bovine mastitis, which results in a decline in milk production, culling of the dairy cow, and increased treatment costs (1, 29). In addition, food-borne S. aureus has become a major public health concern owing to the rapid evolution of resistant lineages (6). A vaccine against S. aureus-induced mastitis in dairy cows would represent a safe and ideal means of preventing and controlling mastitis (27).S. aureus infection is a complex process that involves a series of events, resulting in malfunction or destruction of host tissues. Adherence of the microorganism to host tissues represents a critical first step. Nonadherent bacteria can be readily removed from the host by clearing mechanisms, such as peristalsis and excretion (3, 8). Thus, blocking bacterial adhesion to cells and colonization of the mucosal surface may be the most effective strategy for preventing S. aureus infection (19).S. aureus clumping factor A (ClfA), which is usually covalently anchored to the peptidoglycan of the bacterial cell wall, is an important adhesin and a critical virulence factor. ClfA mediates the binding of S. aureus to fibrinogen on the host cell surface and promotes bacterial invasion into host tissues. An S. aureus clfA mutant displayed reduced virulence in mice (16). Stutzmann et al. (26) showed that introduction of the clfA gene into a less virulent organism, such as Streptococcus gordonii, significantly increased its infective capacity in a mouse endocarditis model. Tuchscherr et al. (30) showed that the passive transfer of antibodies to ClfA protected mice against mastitis. As ClfA does not show genetic polymorphism, it appears to be a suitable component of a novel vaccine against S. aureus infection and the resultant mastitis (2, 23). Josefsson et al. (10) demonstrated that the severity of arthritis was markedly reduced in mice immunized with ClfA. A DNA vaccine that encodes ClfA, as well as the fibronectin-binding motifs of FnBP, delivered twice and boosted once with recombinant fibronectin-binding motifs and ClfA proteins provided partial protection to the mammary gland against staphylococcal mastitis and produced better postchallenge conditions in vaccinated heifers (25). However, a safety concern is that the introduced DNA may be integrated into the host cell chromosomes by insertional mutagenesis (5). To enhance the immune responses induced by ClfA, the potent cytokine interleukin-18 (IL-18) has been used as an adjuvant (32).The fragment of ClfA responsible for its activity lies within binding region A of ClfA (ClfA-A) (14). Hartford et al. (7) localized the fibrinogen-binding activity of ClfA to residues 221 to 559 of region A. Furthermore, the fibrinogen-binding sites P336 and Y338 of clumping factor A are crucial for S. aureus virulence (9). ClfA-A not only promotes bacterial binding to the cell surface but also camouflages S. aureus so as to inhibit phagocytosis (19). In addition, immunization with purified ClfA-A was found to protect against staphylococcus-mediated arthritis (10).In the present study, ClfA-A was expressed and subunit vaccines were prepared as several combinations of recombinant ClfA-A (rClfA-A) and various adjuvants, and these were then evaluated in a BALB/c mouse model of mastitis. The results indicate that a vaccine formulation composed of rClfA-A and an appropriate adjuvant was effective in the prevention of S. aureus-induced mastitis.     

Field Studies of an Attenuated Venezuelan Equine Encephalomyelitis Vaccine (Strain TC-83)

A series of field studies using strain TC-83 attenuated Venezuelan equine encephalomyelitis vaccine in horses was made to determine the rate of seroconversions, the postvaccination viremia, and the possibility of adverse reactions to the vaccine. The rate of seroconversions varied from 50% in one study to 91 and 100% in two others. The highest level of viremia measured was 7 x 10(3) to 8 x 10(3) plaqueforming units per ml. No adverse reactions to the vaccine were observed in any horses, including 42 pregnant mares and their resulting foals.     

Genomic Evolution of Severe Acute Respiratory Syndrome Coronavirus 2 in India and Vaccine Impact

Recent emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and subsequent containment procedures have impacted the world as never seen before. Therefore, there is considerable curiosity about the genome evolution related to the origin, transmission and vaccine impact of this virus. We have analysed genome sequences of SARS-CoV-2 isolated from Indian patients to gain an in-depth understanding of genomic evolution and transmission in India. Phylogenetic analysis and mutation profiling revealed major lineages being evolved by characteristic mutations. As the mutation frequency in spike protein is comparatively lesser, the candidate vaccines expected to have wide coverage worldwide including India.     

Proteomics-Based Identification of Anchorless Cell Wall Proteins as Vaccine Candidates against Staphylococcus aureus

Staphylococcus aureus is an important human pathogen with increasing clinical impact due to the extensive spread of antibiotic-resistant strains. Therefore, development of a protective polyvalent vaccine is of great clinical interest. We employed an intravenous immunoglobulin (IVIG) preparation as a source of antibodies directed against anchorless S. aureus surface proteins for identification of novel vaccine candidates. In order to identify such proteins, subtractive proteome analysis (SUPRA) of S. aureus anchorless cell wall proteins was performed. Proteins reacting with IVIG but not with IVIG depleted of S. aureus-specific opsonizing antibodies were considered vaccine candidates. Nearly 40 proteins were identified by this preselection method using matrix-assisted laser desorption ionization—time of flight analysis. Three of these candidate proteins, enolase (Eno), oxoacyl reductase (Oxo), and hypothetical protein hp2160, were expressed as glutathione S-transferase fusion proteins, purified, and used for enrichment of corresponding immunoglobulin Gs from IVIG by affinity chromatography. Use of affinity-purified anti-Eno, anti-Oxo, and anti-hp2160 antibodies resulted in opsonization, phagocytosis, and killing of S. aureus by human neutrophils. High specific antibody titers were detected in mice immunized with recombinant antigens. In mice challenged with bioluminescent S. aureus, reduced staphylococcal spread was measured by in vivo imaging. The recovery of S. aureus CFU from organs of immunized mice was diminished 10- to 100-fold. Finally, mice immunized with hp2160 displayed statistically significant higher survival rates after lethal challenge with clinically relevant S. aureus strains. Taken together, our data suggest that anchorless cell wall proteins might be promising vaccine candidates and that SUPRA is a valuable tool for their identification.Staphylococcus aureus is an opportunistic, nosocomial, community-acquired pathogen which causes several diseases ranging from minor skin infections to serious life-threatening infections like sepsis, endocarditis, pneumonia, and toxic shock syndrome (30). The rapid emergence of both hospital-associated methicillin (meticillin)-resistant S. aureus (MRSA) and community-acquired MRSA (CA-MRSA) is a major epidemiological problem worldwide (5, 25). A further threatening trend concerning S. aureus infections is the emergence of isolates with resistance to vancomycin, currently the antibiotic of choice against MRSA strains, and also to newly introduced drugs, such as daptomycin and linezolid (47). Hence, it is not surprising that interest in developing alternative approaches to prevent and treat staphylococcal infections has increased in recent years (34, 48).The major effector mechanism of the human immune system against S. aureus infection is comprised of professional phagocytes, such as neutrophils, that ingest and eliminate bacteria (16). However, phagocytosis of S. aureus relies on the opsonization of bacteria by antibodies and complement (7). Recognition of opsonizing antibodies bound to the surface of S. aureus via Fcγ receptors of neutrophils is a prerequisite for induction of the oxidative burst and therefore for killing of the phagocytosed bacteria (23) and induction of a long-term immune response (38). On the other hand, the presence of antistaphylococcal antibodies does not guarantee protection against reinfection. The reason for this apparent discrepancy is still not well understood. However, it was reported recently that antibodies against certain staphylococcal antigens present in healthy donors were missing or underrepresented in patient sera, indicating that antibodies reacting to these antigens are more efficient for induction of phagocytosis and for subsequent elimination of S. aureus than other antibodies (10, 12). Because of this, identification of protective antigens is a crucial step for vaccine development.Until now, most strategies for vaccination against S. aureus, active or passive, concentrated on single-component vaccines based on capsular polysaccharides or well-known virulence factors possessing the LPXTG motif, such as fibronectin binding protein (FnBP), collagen binding protein (CnBP), or clumping factor A (ClfA), as vaccination targets (14, 44). However, despite promising vaccination results obtained with animal models, so far most of the potential vaccines tested in clinical trials have failed to provide significant protection against S. aureus infection (48).It turns out that the efficacy of a monovalent vaccine may be hampered by the functional redundancy of adhesion proteins (17) or the appearance of escape mutants (56). Recently, it has been shown that a multivalent vaccine consisting of four antigenic determinants provides protection against lethal challenge with S. aureus in mice, whereas single-component immunization was much less effective (55). Therefore, identification of novel targets for an effective S. aureus vaccine has repeatedly been recognized as a high priority by experts in this field (20, 28, 34, 40, 43, 48). Indeed, numerous staphylococcal surface proteins predicted to be promising antigenic targets have been identified so far using recently adopted technologies, like proteomics (19, 36, 57) or protein selection methods based on expression libraries (10, 13, 58, 59). Unfortunately, most studies have not provided functional proof that identified proteins are vaccine candidates.Due to the fact that most of the previous experimental vaccine studies concentrated on candidates exhibiting the LPXTG sorting signal, we focused primarily on identification of noncovalently linked, cell wall-associated proteins, so-called anchorless cell wall (ACW) proteins. Proteins belonging to this class possess neither a conserved signal peptide nor an LPXTG motif and were recently recognized as novel virulence factors in gram-positive bacteria (9). Most of these ACW proteins are multifunctional; e.g., they are involved in different metabolic pathways and also in adhesion to extracellular matrix and invasion of host cells. Such proteins cannot be targeted by genome sequence screening due to the lack of conserved epitopes like LPXTG.For identification of new potential vaccine targets among ACW proteins we used intravenous immunoglobulin (IVIG) preparations to avoid limitation of the antibody source (i.e., individual sera). IVIG is a pool of immunoglobulins (Igs) from healthy persons that contains a broad spectrum of opsonizing antibodies against various pathogens, including S. aureus. By employing two-dimensional gel electrophoresis (2-DE), subtractive immunoblotting, and mass spectrometry, we identified several ACW proteins as novel vaccine targets. This method for identification of potential vaccines was called subtractive proteome analysis (SUPRA). The protective activity of antibodies raised against some of the identified proteins was evaluated in vitro and in a murine sepsis model.     

Evaluation of Regulated Delayed Attenuation Strategies for Salmonella enterica Serovar Typhi Vaccine Vectors in Neonatal and Infant Mice

We developed regulated delayed attenuation strategies for Salmonella vaccine vectors. In this study, we evaluated the combination of these strategies in recombinant attenuated Salmonella enterica serovar Typhi and Salmonella enterica serovar Typhimurium vaccine vectors with similar genetic backgrounds in vitro and in vivo. Our goal is to develop a vaccine to prevent Streptococcus pneumoniae infection in newborns; thus, all strains delivered a pneumococcal antigen PspA and the impact of maternal antibodies was evaluated. The results showed that all strains with the regulated delayed attenuated phenotype (RDAP) displayed an invasive ability stronger than that of the S. Typhi vaccine strain, Ty21a, but weaker than that of their corresponding wild-type parental strains. The survival curves of different RDAP vaccine vectors in vitro and in vivo exhibited diverse regulated delayed attenuation kinetics, which was different from S. Typhi Ty21a and the wild-type parental strains. Under the influence of maternal antibody, the persistence of the S. Typhimurium RDAP strain displayed a regulated delayed attenuation trend in nasal lymphoid tissue (NALT), lung, and Peyer''s patches, while the persistence of S. Typhi RDAP strains followed the curve only in NALT. The bacterial loads of S. Typhi RDAP strains were lower in NALT, lung, and Peyer''s patches in mice born to immune mothers than in those born to naive mothers. In accordance with these results, RDAP vaccine strains induced high titers of IgG antibodies against PspA and against Salmonella lipopolysaccharides. Immunization of mothers with S. Typhi RDAP strains enhanced the level of vaginal mucosal IgA, gamma interferon (IFN-γ), and interleukin 4 (IL-4) and resulted in a higher level of protection against S. pneumoniae challenge.     

Comparative Adjuvant Effects of Type II Heat-Labile Enterotoxins in Combination with Two Different Candidate Ricin Toxin Vaccine Antigens

Type II heat-labile enterotoxins (HLTs) constitute a promising set of adjuvants that have been shown to enhance humoral and cellular immune responses when coadministered with an array of different proteins, including several pathogen-associated antigens. However, the adjuvant activities of the four best-studied HLTs, LT-IIa, LT-IIb, LT-IIb_T13I, and LT-IIc, have never been compared side by side. We therefore conducted immunization studies in which LT-IIa, LT-IIb, LT-IIb_T13I, and LT-IIc were coadministered by the intradermal route to mice with two clinically relevant protein subunit vaccine antigens derived from the enzymatic A subunit (RTA) of ricin toxin, RiVax and RVEc. The HLTs were tested with low and high doses of antigen and were assessed for their abilities to stimulate antigen-specific serum IgG titers, ricin toxin-neutralizing activity (TNA), and protective immunity. We found that all four HLTs tested were effective adjuvants when coadministered with RiVax or RVEc. LT-IIa was of particular interest because as little as 0.03 μg when coadministered with RiVax or RVEc proved effective at augmenting ricin toxin-specific serum antibody titers with nominal evidence of local inflammation. Collectively, these results justify the need for further studies into the mechanism(s) underlying LT-IIa adjuvant activity, with the long-term goal of evaluating LT-IIa''s activity in humans.