Comparative evaluation of a vaccine candidate expressing enterotoxigenic Escherichia coli (ETEC) adhesins for colibacillosis with a commercial vaccine using a pig model

In this study, a comparative evaluation of a novel live vaccine candidate expressing enterotoxigenic Escherichia coli (ETEC) fimbriae and a commercial ETEC vaccine was carried out in suckling to weaned piglets. The E. coli K88ab, K88ac, K99, FasA and F41 fimbrial genes were individually inserted into an expression/secretion plasmid, pBP244. These plasmids were subsequently transfected into attenuated Salmonella, which were used as the vaccine candidate. Eighteen pregnant sows and 107 of their piglets were used in this comparative study. All the vaccinated groups of sows and piglets exhibited significantly increased antibody levels relative to specific antigens when compared with those in the unimmunized control. The experimental piglets with the vaccine candidate did not experience diarrhea following challenge with the virulent ETEC strains. However, diarrhea was observed in 36.8% of the piglets in the group immunized with the commercial vaccine and in 50% of the control group after challenge with the ETEC strains. These findings indicate that immunization of sows with the candidate vaccine can effectively protect their young pigs against colibacillosis.     

Immune responses elicited against multiple enterotoxigenic Escherichia coli fimbriae and mutant LT expressed in attenuated Shigella vaccine strains

Shigella and enterotoxigenic Escherichia coli (ETEC) continue to be important causes of diarrheal disease in infants and young children in developing countries and are major etiologic agents of traveler's diarrhea. Since attenuated strains of Shigella have been developed as live oral vaccines against shigellosis, we have adapted these attenuated Shigella strains to serve as carriers of ETEC antigens, thereby constituting a hybrid vaccine. Since protective immunity against ETEC is largely directed against fimbrial antigens (of which there are multiple antigenic types), we have individually expressed four different ETEC fimbriae, including CFA/I, CS2, CS3, and CS4, using deltaguaBA attenuated Shigella vaccine strain CVD 1204 as a prototype live vector. Following mucosal (intranasal) immunization of guinea pigs, serum IgG and mucosal IgA responses were elicited against each fimbrial type. An additional strain was constructed expressing a detoxified version of the human ETEC variant of heat labile toxin (LThK63). Following mucosal immunization of guinea pigs with a mixed inoculum containing five Shigella strains each expressing a different ETEC antigen, immune responses were observed against each ETEC antigen plus the Shigella vector.     

Status of vaccine research and development for enterotoxigenic Escherichia coli

Enterotoxigenic Escherichia coli (ETEC) is one of the most common bacterial causes of diarrhea-associated morbidity and mortality, particularly among infants and young children in developing countries. Still, the true impact on child and traveler health is likely underestimated. There are currently no licensed vaccines for ETEC, but studies indicate high public health impact, cost-effectiveness, and feasibility of immune protection through vaccination. ETEC vaccine development remains a World Health Organization priority. Traditionally, ETEC vaccine development efforts have focused on inducing antitoxin and anticolonization antigen immunity, as studies indicate that antibodies against both antigen types can contribute to protection and thus have potential for vaccines. Leading cellular vaccine candidates are ETVAX (a mixture of four inactivated strains) and ACE527 (a mixture of three live attenuated strains), both of which have been found to be safe and immunogenic in Phase 1/2 trials. ETVAX is the furthest along in development with descending-age studies already underway in Bangladesh. Other ETEC vaccine candidates based on protein subunits, toxoids (both LT and ST), or novel, more broadly conserved ETEC antigens are also under development. Of these, a protein adhesin-based subunit approach is the most advanced. Impact and economic models suggest favorable vaccine cost-effectiveness, which may help expand market interest in ETEC vaccines. Combination vaccine formulations may help improve the economic case for development and use, and better point-of-care diagnostics will help to raise awareness of the true health burden of ETEC and highlight the potential public health benefit of ETEC vaccine introduction. Better diagnostics and vaccine demand forecasting will also improve vaccine development financing and support accelerated uptake once a licensed vaccine becomes available.     

Development and preclinical evaluation of safety and immunogenicity of an oral ETEC vaccine containing inactivated E. coli bacteria overexpressing colonization factors CFA/I,CS3, CS5 and CS6 combined with a hybrid LT/CT B subunit antigen,administered alone and together with dmLT adjuvant

A first-generation oral inactivated whole-cell enterotoxigenic Escherichia coli (ETEC) vaccine, comprising formalin-killed ETEC bacteria expressing different colonization factor (CF) antigens combined with cholera toxin B subunit (CTB), when tested in phase III studies did not significantly reduce overall (generally mild) ETEC diarrhea in travelers or children although it reduced more severe ETEC diarrhea in travelers by almost 80%. We have now developed a novel more immunogenic ETEC vaccine based on recombinant non-toxigenic E. coli strains engineered to express increased amounts of CF antigens, including CS6 as well as an ETEC-based B subunit protein (LCTBA), and the optional combination with a nontoxic double-mutant heat-labile toxin (LT) molecule (dmLT) as an adjuvant.     

U-Omp19 from Brucella abortus increases dmLT immunogenicity and improves protection against Escherichia coli heat-labile toxin (LT) oral challenge

Acute diarrhea disease caused by bacterial infections is a major global health problem. Enterotoxigenic Escherichia coli (ETEC) is one of the top causes of diarrhea-associated morbidity and mortality in young children and travelers to low-income countries. There are currently no licensed vaccines for ETEC. Induction of immunity at the site of entry of the bacteria is key to prevent infection. Current approaches to ETEC vaccines include a less toxic mutant form of E. coli heat-labile toxin (double-mutant heat-labile enterotoxin -dmLT-) with both antigenic and immunostimulatory properties. U-Omp19 is a protease inhibitor from Brucella spp. with immunostimulatory properties that has been used as oral adjuvant. In this work, we use U-Omp19 as adjuvant in an oral vaccine formulation against ETEC containing dmLT in outbred and inbred mice. To evaluate antigen dose sparing by U-Omp19 three different immunization protocols with three different doses of dmLT were evaluated. We demonstrated that U-Omp19 co-delivery increases anti-LT IgA in feces using a mid-dose of dmLT following a prime-boost protocol (after one or two boosts). Oral immunization with U-Omp19 induced protection against LT challenge when co-formulated with dmLT in CD-1 and BALB/c mice. Indeed, there was a significant increase in anti-LT IgG and IgA avidity after a single oral administration of dmLT plus U-Omp19 in comparison with dmLT delivered alone. Interestingly, sera from dmLT plus U-Omp19 vaccinated mice significantly neutralize LT effect on intestine inflammation in vivo compared with sera from the group immunized with dmLT alone. These results demonstrate the adjuvant capacity of U-Omp19 to increase dmLT immunogenicity by the oral route and support its use in an oral subunit vaccine formulation against ETEC.     

Protection from Staphylococcus aureus mastitis associated with poly-N-acetyl β-1,6 glucosamine specific antibody production using biofilm-embedded bacteria

Staphylococcus aureus vaccines based on bacterins surrounded by slime, surface polysaccharides coupled to protein carriers and polysaccharides embedded in liposomes administered together with non-biofilm bacterins confer protection against mastitis. However, it remains unknown whether protective antibodies are directed to slime-associated known exopolysaccharides and could be produced in the absence of bacterin immunizations. Here, a sheep mastitis vaccination study was carried out using bacterins, crude bacterial extracts or a purified exopolysaccharide from biofilm bacteria delivered in different vehicles. This polysaccharide reacted specifically with antibodies to poly-N-acetyl-β-1,6-glucosamine (PNAG) and not with antibodies to other capsular antigens or bacterial components. Following intra-mammary challenge with biofilm-producing bacteria, antibody production against the polysaccharide, milk bacterial counts and mastitis lesions were determined. Bacterins from strong biofilm-producing bacteria triggered the highest production of antibodies to PNAG and conferred the highest protection against infection and mastitis, compared with weak biofilm-producing bacteria and non-cellular inocula. Thus, bacterins from strong biofilm bacteria, rather than purified polysaccharide, are proposed as a cost-efficient vaccination against S. aureus ruminant mastitis.     

Immune responses of a chimaeric protein vaccine containing Mycoplasma hyopneumoniae antigens and LTB against experimental M. hyopneumoniae infection in pigs

A recombinant chimaeric protein containing three Mycoplasma hyopneumoniae antigens (C-terminal portion of P97, heat shock protein P42, and NrdF) fused to an adjuvant, the B subunit of heat-labile enterotoxin of Escherichia coli (LTB), was used to immunize pigs against enzootic pneumonia. The systemic and local immune responses, as well as the efficacy of the chimaeric protein in inducing protection against experimental M. hyopneumoniae infection were evaluated. In total, 60 male piglets, purchased from a M. hyopneumoniae-free herd, at 4 weeks of age were randomly allocated to six different experimental groups of 10 animals each: recombinant chimaeric protein by intramuscular (IM) (1) or intranasal (IN) (2) administration, commercial bacterin by IM administration (3), and the adjuvant LTB by IM (4, control group A) or IN (5, control group B) administration. All groups were immunized at 24 and 38 days of age and challenged at 52 days of age. The sixth group that was not challenged was used as the negative control (IN [n = 5] or IM [n = 5] administration of the LTB adjuvant). Compared with the non-challenged group, administration of the chimaeric protein induced significant (P < 0.05) IgG and IgA responses against all individual antigens present in the chimaera, but it could not confer a significant protection against M. hyopneumoniae infection in pigs. This lack of effectiveness points towards the need for further studies to improve the efficacy of this subunit-based vaccine approach.     

Analysis of strategies to successfully vaccinate infants in developing countries against enterotoxigenic E. coli (ETEC) disease

Enterotoxigenic Escherichia coli (ETEC) is the most common bacterial cause of diarrhoea in the world, annually affecting up to 400,000,000 children under 5 years of age living in developing countries (DCs). Although ETEC possesses numerous antigens, the relatively conserved colonization factor (CF) antigens and the heat labile enterotoxin (LT) have been associated with protection and most vaccine candidates have exploited these antigens. A safe and effective vaccine against ETEC is a feasible goal as supported by the acquisition of protective immunity. The success of an ETEC vaccine targeting infants and children in DCs will depend on a combination of maximally antigenic vaccine preparations and regimens for their delivery which will produce optimal immune responses to these antigens. Vaccine candidates having a high priority for accelerated development and clinical testing for eventual use in infants would include inactivated ETEC or Shigella hybrids expressing ETEC antigens as well as attenuated ETEC strains which express the major CF antigens and LT toxin B-subunit, as well as attenuated Shigella, Vibrio cholerae and Salmonella typhi hybrids engineered to deliver antigens of ETEC. Candidates for an ETEC vaccine would have to meet the minimal requirement of providing at least 50% protection against severe disease in DCs during the first 2 years of life. The critical roadblock to achieving this goal has not been the science as much as the lack of a sufficiently funded and focused effort to bring it to realization. However, a Product Development Partnership to overcome this hurdle could accelerate the time lines towards when control of ETEC disease in DCs is substantially closer.     

Evaluation of recombinant Mycoplasma hyopneumoniae P97/P102 paralogs formulated with selected adjuvants as vaccines against mycoplasmal pneumonia in pigs

Pig responses to recombinant subunit vaccines containing fragments of eight multifunctional adhesins of the Mycoplasma hyopneumoniae (Mhp) P97/P102 paralog family formulated with Alhydrogel^® or Montanide™ Gel01 were compared with a commercial bacterin following experimental challenge. Pigs, vaccinated intramuscularly at 9, 12 and 15 weeks of age with either of the recombinant formulations (n = 10 per group) or Suvaxyn^® M. hyo (n = 12), were challenged with Mhp strain Hillcrest at 17 weeks of age. Unvaccinated, challenged pigs (n = 12) served as a control group. Coughing was assessed daily. Antigen-specific antibody responses were monitored by ELISA in serum and tracheobronchial lavage fluid (TBLF), while TBLF was also assayed for cytokine responses (ELISA) and bacterial load (qPCR). At slaughter, gross and histopathology of lungs were quantified and damage to epithelial cilia in the porcine trachea was evaluated by scanning electron microscopy. Suvaxyn^® M. hyo administration induced significant serological responses against Mhp strain 232 whole cell lysates (wcl) and recombinant antigen F3_P216, but not against the remaining vaccine subunit antigens. Alhydrogel^® and Montanide™ Gel01-adjuvanted antigen induced significant antigen-specific IgG responses, with the latter adjuvant eliciting comparable Mhp strain 232 wcl specific IgG responses to Suvaxyn^® M. hyo. No significant post-vaccination antigen-specific mucosal responses were detected with the recombinant vaccinates. Suvaxyn^® M. hyo was superior in reducing clinical signs, lung lesion severity and bacterial load but the recombinant formulations offered comparable protection against cilial damage. Lower IL-1β, TNF-α and IL-6 responses after challenge were associated with reduced lung lesion severity in Suvaxyn^® M. hyo vaccinates, while elevated pathology scores in recombinant vaccinates corresponded to cytokine levels that were similarly elevated as in unvaccinated pigs. This study highlights the need for continued research into protective antigens and vaccination strategies that will prevent Mhp colonisation and establishment of infection.     

Evaluation of a conjugate vaccine platform against enterotoxigenic Escherichia coli (ETEC), Campylobacter jejuni and Shigella

Enterotoxigenic Escherichia coli (ETEC), Campylobacter jejuni (CJ), and Shigella sp. are major causes of bacterial diarrhea worldwide, but there are no licensed vaccines against any of these pathogens. Most current approaches to ETEC vaccines are based on recombinant proteins that are involved in virulence, particularly adhesins. In contrast, approaches to Shigella and CJ vaccines have included conjugate vaccines in which Shigella lipopolysaccharides (LPS) or CJ capsule polysaccharides are chemically conjugated to proteins. We have explored the feasibility of developing a multi-pathogen vaccine by using ETEC proteins as conjugating partners for CJ and Shigella polysaccharides. We synthesized three vaccines in which two CJ polysaccharides were conjugated to two recombinant ETEC adhesins based on CFA/I (CfaEB) and CS6 (CssBA), and LPS from Shigella flexneri was also conjugated to CfaEB. The vaccines were immunogenic in mice as monovalent, bivalent and trivalent formulations. Importantly, functional antibodies capable of inducing hemaglutination inhibition (HAI) of a CFA/I expressing ETEC strain were induced in all vaccines containing CfaEB. These data suggest that conjugate vaccines could be a platform for a multi-pathogen, multi-serotype vaccine against the three major causes of diarrheal disease worldwide.     

Identification of protective antigens by RNA interference for control of the lone star tick, Amblyomma americanum

The lone star tick, Amblyomma americanum, vectors pathogens of emerging diseases of humans and animals in the United States. Currently, measures are not available for effective control of A. americanum infestations. Development of vaccines directed against tick proteins may reduce tick infestations and the transmission of tick-borne pathogens. However, the limiting step in tick vaccine development has been the identification of tick protective antigens. Herein, we report the application of RNA interference (RNAi) for screening an A. americanum cDNA library for discovery of tick protective antigens that reduce tick survival and weights after feeding. Four cDNA clones, encoding for putative threonyl-tRNA synthetase (2C9), 60S ribosomal proteins L13a (2D10) and L13e (2B7), and interphase cytoplasm foci protein 45 (2G7), were selected for vaccine studies in cattle, along with subolesin, a tick protective protein identified previously. In vaccinated cattle, an overall efficacy (E) > 30% was obtained when considering the vaccine effect on both nymphs and adults, but only 2D10, 2G7 and subolesin affected both tick stages. The highest efficacy of control for adult ticks (E > 55%) was obtained in cattle vaccinated with recombinant 2G7 or subolesin. These collective results demonstrated the feasibility of developing vaccines for the control of lone star tick infestations. The use of RNAi for identification of tick protective antigens proved to be a rapid and cost-effective tool for discovery of candidate vaccine antigens, and this approach could likely be applied to other parasites of veterinary and medical importance.     

Evaluation of 238 antigens of Leptospira borgpetersenii serovar Hardjo for protection against kidney colonisation

Leptospirosis is a zoonotic disease affecting animals and humans worldwide. Leptospiral infection in cattle can cause reproductive failure and reduced weight gain, and importantly, infection represents a significant disease risk for farmers. Current bacterin vaccines offer protection that is short-lived and restricted at best to related serovars. The development of protective vaccines that stimulate immunity across multiple leptospiral serovars would therefore be advantageous. This study used a reverse vaccinology approach to evaluate a set of Leptospira borgpetersenii proteins in the hamster infection model. The L. borgpetersenii serovar Hardjo strain L550 genome sequence was analysed and genes encoding 262 predicted outer membrane or secreted proteins were selected. From this list, 238 proteins or protein fragments were successfully expressed and purified; 28 proteins (12%) were soluble, while the remaining 210 proteins (88%) were insoluble and purified under denaturing conditions. Proteins were mixed into 48 pools of up to five each and tested for protection against infection as assessed by renal colonisation in the hamster model of infection. None of the pools of antigens protected the hamsters against infection, despite a detectable antibody response being mounted against the majority of proteins (71%). This study is the first large scale evaluation of individual leptospiral proteins for ability to induce a protective immune response in the hamster infection model. It thus constitutes an important reference of protein immunogenicity and non-protective antigens that should be consulted before embarking on any future subunit vaccine experiments.     

Oral immunization of a live attenuated Escherichia coli strain expressing a holotoxin-structured adhesin–toxoid fusion (1FaeG-FedF-LTA2:5LTB) protected young pigs against enterotoxigenic E. coli (ETEC) infection

ETEC strains expressing K88 (F4) or F18 fimbriae and enterotoxins are the predominant cause of porcine post-weaning diarrhea (PWD). PWD continues causing significant economic losses to swine producers worldwide. Vaccines effectively protecting against PWD are needed. Our recent study revealed that a tripartite adhesin–toxin monomer (FaeG-FedF-LT_A2–B) elicited protective antibodies. In this study, we constructed a new adhesin–toxoid fusion, expressed it as a 1A:5B holotoxin-structured antigen (1FaeG-FedF-LT_192A2:5LT_B) in an avirulent Escherichia coli strain, and evaluated its vaccine potential in pig challenge studies. Piglets orally inoculated with this live strain showed no adverse effects but developed systemic and mucosal antibodies that neutralized cholera toxin and inhibited adherence of K88 and F18 fimbriae in vitro. Moreover, the immunized piglets, when were challenged with ETEC strain 3030-2 (K88ac/LT/STb), had significant fewer bacteria colonized at small intestines and did not develop diarrhea; whereas the control piglets developed severe diarrhea and died. These results indicated the 1FaeG-FedF-LT_192A2:5LT_B fusion antigen induced protective antiadhesin and antitoxin immunity in pigs, and suggested a live attenuated vaccine can be potentially developed against porcine ETEC diarrhea. Additionally, presenting antigens in a holotoxin structure to target host local mucosal immunity can be used in vaccine development against other enteric diseases.     

Modulation of inter-vaccination interval to avoid antigenic competition in multivalent footrot (Dichelobacter nodosus) vaccines in sheep

Virulent footrot is a significant disease of sheep in most sheep farming countries; a strain/serogroup of the anaerobic bacterium Dichelobacter nodosus is the essential transmitting agent. Commercial multivalent footrot vaccines containing nine fimbrial serogroups (A through I) of D. nodosus produce relatively low and short term antibody responses due to antigenic competition, in contrast to higher and longer responses provided by monovalent or bivalent vaccines. The latter were important components of successful eradication programs for endemic footrot caused by either one or two serogroups of D. nodosus in Nepal, Bhutan, and several flocks in Australia. However, the presence of up to six serogroups in some Australian flocks and the use of an annual bivalent vaccination regime to progressively eradicate serogroups would require a long term program. In this study we report the results of a sequential vaccination trial testing different time intervals between different bivalent vaccinations. Intervals of 12, 9, 6, 3 and 0 months were tested. The 1st vaccination was with recombinant fimbrial antigens for serogroups A and B while the 2nd vaccination was with D and E. There were no significant differences between the antibody responses for time intervals of 3, 6, 9 and 12 months whereas there was a reduced response when sheep were vaccinated with two bivalent vaccines (four antigens) concurrently, indicating antigenic competition. Therefore an inter-vaccination interval of 3 months can be applied between two different bivalent vaccines without detrimental impact on the humoral immune responses to the various fimbrial antigens of D. nodosus. These results could have wider applications in vaccination against diseases caused by multivalent or multistrain microbes.     

Immunogenic potential of latency associated antigens against Mycobacterium tuberculosis

Tuberculosis remains a great health threat to the world among infectious diseases particularly with the advent of human immunodeficiency virus and emergence of drug resistant strains. In the light of the inconsistent efficacy imparted by the only currently available pre-exposure vaccine bacillus Calmette–Guerin BCG, the development of an improved TB vaccine is a very high international research priority. Vaccine candidates currently in clinical trials are also pre-exposure vaccines that aim to prevent active tuberculosis during an individual's lifetime. According to World Health Organization approximately a third of the world's population is latently infected with Mycobacterium tuberculosis. Dormancy or latency of Mycobacteria is associated with the formation of granuloma with poorly perfused interior leading to expression of genes which help them survive in this hostile environment. A group of about 50 genes belonging to the DosR regulon also known as latency antigens are expressed by Mycobacteria when they are persisting in the immuno-competent host. An understanding of the immunological effects produced by products of these latency induced genes may help in making a more potent vaccine. Incorporation of latency antigens into improved (live or subunit) vaccines may enhance the impact of these vaccines in which BCG priming can be followed by multisubunit protein boosting. These vaccines could act as post exposure vaccines for containment and prevention of latent TB activation. This heterologous boosting of BCG-primed immunity will be able to stimulate the known immune correlates of protective immunity against M. tuberculosis i.e. T_H1 cells (CD4⁺ and CD8⁺ T cells) mediated immune responses with cytokines such as IFN-γ and TNF-α⋅ In our review we have analysed and compared the immunogenic potential of various latency-associated antigens of the DosR regulon in line with the current strategy of developing a recombinant post exposure booster vaccine.     

Parenteral administration of attenuated Salmonella Typhimurium ΔznuABC is protective against salmonellosis in piglets

A major cause of salmonellosis in humans is the contamination of pork products. Infection in pigs can be controlled using bio-security programs, but they are not sufficient in countries where a high level of infection is recorded. In this context, the use of vaccines can represent a valid supplementary method of control. Recently, we have demonstrated that an attenuated strain of Salmonella enterica serovar Typhimurium (Salmonella Typhimurium ΔznuABC) is protective against systemic and enteric salmonellosis in mouse and pig infection models, candidating this strain as an oral attenuated vaccine. In this study, we compared the efficacy of this attenuated Salmonella Typhimurium strain when administered orally or parenterally. Furthermore, in order to reproduce a pseudo-natural infection model, vaccinated pigs were allocated in the same pen with animals shedding virulent Salmonella Typhimurium. Animals were monitored weekly after vaccination and contact with infected piglets. Diarrhea and ataxia were recorded and Salmonella shedding was tested individually through bacterial culture. After four weeks of cohousing, piglets were euthanized, after which lymph nodes reactivity and gross lesions of the gut sections were scored at necropsy. Organs were submitted to microbiological and histological analyses.     

Protective efficacy of whole-cell inactivated Leptospira vaccines made using virulent or avirulent strains in a hamster model

Whole-cell inactivated vaccines remain the only licensed vaccines used to control human and animal leptospirosis worldwide. Although they are protective against lethal infections, the efficacy of these vaccines has been divergent. The manufacturing process often involves the use of standard bacterial strains subjected to serial in vitro passages, with a risk of loss of virulence, and may affect the immunogenicity and consequently decrease protection. Thus, the objective of this study was to perform a comparative analysis of the efficacy of in-house bacterins produced with standard (avirulent) and virulent strains. Hamsters were immunized with killed bacteria produced using avirulent and virulent strains of L. interrogans serovars Copenhageni and Canicola. Vaccine efficacy was determined in terms of protection against lethal homologous or heterologous challenges. The results showed that immunization with both avirulent and virulent Canicola strains resulted in 100% protection against homologous challenge. Conversely, Copenhageni bacterins produced using an avirulent strain conferred only 25–37.5% protection against homologous challenge (P > 0.05), while virulent Copenhageni bacterin conferred 100% protection (P < 0.001). A single vaccine dose was sufficient to induce protection, and administration of a prime boost significantly reduced the bacterial load in the kidneys and improved the humoral immune response to the virulent Copenhageni strain. These findings suggest that the maintenance of virulent strains in bacterin formulations is essential for improving the immunogenicity and efficacy of leptospirosis vaccines.     

Vaccination with EtpA glycoprotein or flagellin protects against colonization with enterotoxigenic Escherichia coli in a murine model

Enterotoxigenic Escherichia coli (ETEC) remain a leading cause diarrheal illness, prompting a search for vaccine targets that led to the recent discovery of EtpA, a secreted adhesin of ETEC that acts by bridging flagella and host cells. In a murine model, immunization with recombinant EtpA glycoprotein inhibited colonization by two EtpA-producing human ETEC strains, H10407 and E24377A. In addition, vaccination with recombinant flagellin (serotype H11) generated antibodies that specifically recognized the tips of flagella from E24377A expressing a heterologous flagellar serotype (H28) and afforded significant protection against colonization. EtpA and/or flagellin could be valuable subunit antigens in the formulation of a broadly protective ETEC vaccine.     

Protective efficacy of a lipid antigen vaccine in a guinea pig model of tuberculosis

The bacillus Calmette Guérin (BCG) vaccine, the only licensed vaccine against TB, displays partial and variable efficacy, thus making the exploitation of novel vaccination strategies a major priority. Most of the current vaccines in pre-clinical or clinical development are based on the induction of T cells recognizing protein antigens. However, a large number of T cells specific for mycobacterial lipids are induced during infection, suggesting that lipid-based vaccines might represent an important component of novel sub-unit vaccines. Here, we investigated whether immunization with defined mycobacterial lipid antigens induces protection in guinea pigs challenged with M. tuberculosis. Two purified mycobacterial lipid antigens, the diacylated sulfoglycolipids (Ac₂SGL) and the phosphatidyl-myo-inositol dimannosides (PIM₂) were formulated in biophysically characterized liposomes made of dimethyl-dioctadecyl-ammonium (DDA) and synthetic trehalose 6,6′-dibehenate (TDB). In three protection trials, a reduction of bacterial load in the spleen of inoculated animals was consistently observed compared to the unvaccinated group. Moreover, a reduction in the number of lesions and severity of pathology was detected in the lungs and spleen of the lipid vaccine group compared to unvaccinated controls. As the degree of protection achieved is similar to that observed using protein antigens in the same guinea pig model, these promising results pave the way to future investigations of lipid antigens as subunit vaccines.     

Antigen engineering can play a critical role in the protective immunity elicited by Yersinia pestis DNA vaccines

The use of a DNA immunization approach to deliver protective antigens against Yersinia pestis (Y. pestis) has been successful in previously reported studies. In the current study, the gene designs for V and F1, two well-studied virulent factors serving as main targets for vaccine development, were altered to explore additional options in hopes of improving the protective immunity of DNA vaccines expressing these two antigens. Compared to the wild type V gene DNA vaccines, the use of codon optimized V gene sequences was effective in improving the antigen expression, titers of anti-V antibody responses, and survival against a mucosal lethal challenge. For the F1 DNA vaccine, removal of the N-terminal hydrophobic region was able to improve protective immunity. However, adding a mammalian signal peptide sequence to F1 actually led to reduced protection despite it inducing slightly higher anti-F1 antibody responses. The F1 gene can be fused with a gene coding for YscF, a newly confirmed partial protective antigen for Y. pestis, to produce DNA vaccines that express fused F1 and YscF antigens. One design, in particular, that had YscF fused to the downstream sequence of F1, produced better protection than separate F1 or YscF DNA vaccines, suggesting a potential synergistic effect between these two antigens. Findings from the above studies indicated that there are multiple approaches to optimize the protective immunity for plague DNA vaccines. Most importantly, proper antigen engineering to produce optimal antigen gene inserts in DNA vaccines can clearly play a major role in the future designs of a wide range of DNA vaccines.