Yersinia pestis is one of the world's most virulent human pathogens. Inhalation of this Gram-negative bacterium causes pneumonic plague, a rapidly progressing and usually fatal disease. Extensively antibiotic-resistant strains of Y. pestis exist and have significant potential for exploitation as agents of terrorism and biowarfare. Subunit vaccines comprised of the Y. pestis F1 and LcrV proteins are well-tolerated and immunogenic in humans but cannot be tested for efficacy, because pneumonic plague outbreaks are uncommon and intentional infection of humans is unethical. In animal models, F1/LcrV-based vaccines protect mice and cynomolgus macaques but have failed, thus far, to adequately protect African green monkeys. We lack an explanation for this inconsistent efficacy. We also lack reliable correlate assays for protective immunity. These deficiencies are hampering efforts to improve vaccine efficacy. Here, I review the immunology of pneumonic plague, focusing on evidence that humoral and cellular defense mechanisms collaborate to defend against pulmonary Y. pestis infection.
Yersinia pestis; phagocytes; neutrophils; macrophages; antibodies; humoral immunity; cellular immunity; vaccine
A number of studies have clearly demonstrated that flagellin is a potent adjuvant that promotes robust immune responses when it is given with a protein antigen. In view of the potential biological and practical benefits of a recombinant protein vaccine composed of a single fusion protein containing flagellin and antigen, we have evaluated the efficacy of a fusion protein composed of flagellin and two protective antigens of Yersinia pestis (F1 and V) in eliciting protection against respiratory challenge with Y. pestis. Flagellin-F1-V was produced and purified in high yield under good manufacturing practices conditions. The fusion protein retains full Toll-like receptor 5-stimulating activity in vitro. Using a prime-boost immunization protocol, we found that flagellin-F1-V elicits robust antigen-specific humoral immunity in mice and two species of nonhuman primates. Immune mice were fully protected against intranasal challenge with 150 mean tolerated doses of Y. pestis CO92. In immune mice, the bacteria were completely cleared within 3 days after challenge. Flagellin-F1-V exhibited full stability for at least 297 days at 4°C and at least 168 days at 25°C. At between 29 and 84 days at 37°C, the protein exhibited a loss of biological activity that appeared to be associated with a substantial change in protein diameter, possibly due to oligomerization. On the basis of our results, we believe that flagellin-F1-V is an outstanding candidate for evaluation in studies with humans.
There is a need to develop effective countermeasures for Yersinia pestis, the etiologic agent of plague and a potential bioterrorism agent. Salmonella and Shigella spp. deleted in the guaBA genes involved in guanine biosynthesis have been shown to be attenuated in vivo. In this study, we sought to determine whether deletion of the guaBA operon would render Y. pestis auxotrophic for guanine and avirulent; such a strain could serve as a live attenuated plague vaccine candidate. A Y. pestis guaBA mutant was generated by specific deletion of a segment of the guaBA operon, producing a guanine auxotroph that was highly attenuated in a mouse model of Y. pestis infection. Furthermore, mice vaccinated with a single dose of 7×104 CFU via the intravenous route were fully protected against subsequent lethal challenge with the Y. pestis parental strain. These findings identify guaBA as a target for deletion to generate a live attenuated plague vaccine.
Yersinia pestis; plague; guanine; mutant; live attenuated vaccines
Presently there is a significant effort to develop and evaluate vaccines and antibiotics against the potential bioterrorism agent Yersinia pestis. The animal models used to test these countermeasures involve the deposition of small particles within the lung. However, deliberate aerosol release of Y. pestis will generate both small and large inhalable particles. We report in this study that the pathogenesis patterns of plague infections caused by the deposition of 1- and 12-μm-particle aerosols of Y. pestis in the lower and upper respiratory tracts (URTs) of mice are different. The median lethal dose for 12-μm particles was 4.9-fold greater than that for 1-μm particles. The 12-μm-particle infection resulted in the degradation of the nasal mucosa and nasal-associated lymphoid tissue (NALT) plus cervical lymphadenopathy prior to bacteremic dissemination. Lung involvement was limited to secondary pneumonia. In contrast, the 1-μm-particle infection resulted in primary pneumonia; in 40% of mice, the involvement of NALT and cervical lymphadenopathy were observed, indicating entry via both URT lymphoid tissues and lungs. Despite bacterial deposition in the gastrointestinal tract, the involvement of Peyer's patches was not observed in either infection. Although there were major differences in pathogenesis, the recombinant F1 and V antigen vaccine and ciprofloxacin protected against plague infections caused by small- and large-particle aerosols.
Pneumonic plague is a highly virulent infectious disease with 100% mortality rate, and its causative organism Yersinia pestis poses a serious threat for deliberate use as a bioterror agent. Currently, there is no FDA approved vaccine against plague. The polymeric bacterial capsular protein F1, a key component of the currently tested bivalent subunit vaccine consisting, in addition, of low calcium response V antigen, has high propensity to aggregate, thus affecting its purification and vaccine efficacy. We used two basic approaches, structure-based immunogen design and phage T4 nanoparticle delivery, to construct new plague vaccines that provided complete protection against pneumonic plague. The NH2-terminal β-strand of F1 was transplanted to the COOH-terminus and the sequence flanking the β-strand was duplicated to eliminate polymerization but to retain the T cell epitopes. The mutated F1 was fused to the V antigen, a key virulence factor that forms the tip of the type three secretion system (T3SS). The F1mut-V protein showed a dramatic switch in solubility, producing a completely soluble monomer. The F1mut-V was then arrayed on phage T4 nanoparticle via the small outer capsid protein, Soc. The F1mut-V monomer was robustly immunogenic and the T4-decorated F1mut-V without any adjuvant induced balanced TH1 and TH2 responses in mice. Inclusion of an oligomerization-deficient YscF, another component of the T3SS, showed a slight enhancement in the potency of F1-V vaccine, while deletion of the putative immunomodulatory sequence of the V antigen did not improve the vaccine efficacy. Both the soluble (purified F1mut-V mixed with alhydrogel) and T4 decorated F1mut-V (no adjuvant) provided 100% protection to mice and rats against pneumonic plague evoked by high doses of Y. pestis CO92. These novel platforms might lead to efficacious and easily manufacturable next generation plague vaccines.
Plague caused by Yersinia pestis is a deadly disease that wiped out one-third of Europe's population in the 14th century. The organism is listed by the CDC as Tier-1 biothreat agent, and currently, there is no FDA-approved vaccine against this pathogen. Stockpiling of an efficacious plague vaccine that could protect people against a potential bioterror attack has been a national priority. The current vaccines based on the capsular antigen (F1) and the low calcium response V antigen, are promising against both bubonic and pneumonic plague. However, the polymeric nature of F1 with its propensity to aggregate affects vaccine efficacy and generates varied immune responses in humans. We have addressed a series of concerns and generated mutants of F1 and V, which are completely soluble and produced in high yields. We then engineered the vaccine into a novel delivery platform using the bacteriophage T4 nanoparticle. The nanoparticle vaccines induced robust immunogenicity and provided 100% protection to mice and rats against pneumonic plague. These highly efficacious new generation plague vaccines are easily manufactured, and the potent T4 platform which can simultaneously incorporate antigens from other biothreat or emerging infectious agents provides a convenient way for mass vaccination of humans against multiple pathogens.
Immune senescence in the elderly results in decreased immunity with a concomitant increase in susceptibility to infection and diminished efficacy of vaccination. Nonhuman primate (NHP) models have proven critical for testing of vaccines and therapeutics in the general population, but a model using old animals has not been established. Towards that end, immunity to LcrV, a protective antigen from Yersinia pestis, was tested in young and old baboons. Surprisingly, there was no age-associated loss in immune competence; LcrV elicited high-titer, protective antibody responses in the older individuals. The primary responses in the younger baboons were lower, but they did show boosting upon secondary immunization to the levels achieved in the old animals. The LcrV antigen was also tested in mice and, as expected, age-associated loss of immunity was seen; older animals responded with lower titer antibodies and as a result, were more susceptible to Yersinia challenge. Thus, although age-related loss in immune function has been observed in humans, rodents and some nonhuman primates, baboons appear to be unusual; they age without losing immune competence.
Other animals (Nonhuman primates); Vaccination; Bacterial Infection
Inhalation of Yersinia pestis causes pneumonic plague, which rapidly progresses to death. A previously licensed killed whole-cell vaccine is presently unavailable due to its reactogenicity and inconclusive evidence of efficacy. The present study now shows that vaccination intranasally (i.n.) with inactivated Y. pestis CO92 (iYp) adjuvanted with interleukin-12 (IL-12) followed by an i.n. challenge with a lethal dose of Y. pestis CO92 prevented bacterial colonization and protected 100% of mice from pneumonic plague. Survival of the vaccinated mice correlated with levels of systemic and lung antibodies, reduced pulmonary pathology and proinflammatory cytokines, and the presence of lung lymphoid cell aggregates. Protection against pneumonic plague was partially dependent upon Fc receptors and could be transferred to naïve mice with immune mouse serum. On the other hand, protection was not dependent upon complement, and following vaccination, depletion of CD4 and/or CD8 T cells before challenge did not affect survival. In summary, the results demonstrate the safety, immunogenicity, and protective efficacy of i.n. administered iYp plus IL-12 in a mouse model of pneumonic plague.
A flea-to-mouse transmission model was developed for use in testing new candidate vaccines for the ability to protect against flea-borne plague. The model was used to evaluate a recombinant fusion protein vaccine consisting of the Yersinia pestis F1 and V antigens. After one to three challenges with Y. pestis-infected fleas, 14 of 15 unvaccinated control mice developed plague, with an average septicemia level of 9.2 × 108 Y. pestis CFU/ml. None of 15 vaccinated mice developed the disease after similar challenges, and serological testing indicated that transmitted bacteria were eliminated by the immune system before extensive replication and systemic infection could occur. The transmission and development of disease in control mice correlated with the number of bites by blocked fleas but not with the total number of fleabites. The model provides a means to directly assess the efficacy of new vaccines to prevent naturally acquired bubonic plague and to study events at the vector-host interface that lead to dissemination and disease.
Despite many decades of intensive studies of Yersinia pestis, the causative agent of plague, there is no safe and efficient vaccine against this devastating disease. A recently developed F1/V subunit vaccine candidate, which relies mainly on humoral immunity, showed promising results in animal studies; however, its efficacy in humans still has to be carefully evaluated. In addition, those developing next-generation plague vaccines need to pay particular attention to the importance of eliciting cell-mediated immunity. In this review, we analyzed the current progress in developing subunit, DNA and live carrier platforms of delivery by bacterial and viral vectors, as well as approaches for controlled attenuation of virulent strains of Y. pestis.
Plague; protective antigens; vaccine; Yersinia pestis
Yersinia pestis, the highly virulent agent of plague, is a biological weapon. Strategies that prevent plague have been sought for centuries, and immunization with live, attenuated (nonpigmented) strains or subunit vaccines with F1 (Caf1) antigen is considered effective. We show here that immunization with live, attenuated strains generates plague-protective immunity and humoral immune responses against F1 pilus antigen and LcrV. Y. pestis variants lacking caf1 (F1 pili) are not only fully virulent in animal models of bubonic and pneumonic plague but also break through immune responses generated with live, attenuated strains or F1 subunit vaccines. In contrast, immunization with purified LcrV, a protein at the tip of type III needles, generates protective immunity against the wild-type and the fully virulent caf1 mutant strain, in agreement with the notion that LcrV can elicit vaccine protection against both types of virulent plague strains.
A recombinant vaccine (rF1V) is being developed for protection against pneumonic plague. This study was performed to address essential data elements to establish a well-characterized Swiss Webster mouse model for licensing the rF1V vaccine using the FDA's Animal Rule. These elements include the documentation of challenge material characteristics, aerosol exposure parameters, details of the onset and severity of clinical signs, pathophysiological response to disease, and relevance to human disease. Prior to animal exposures, an evaluation of the aerosol system was performed to determine and understand the variability of the aerosol exposure system. Standardized procedures for the preparation of Yersinia pestis challenge material also were developed. The 50% lethal dose (LD50) was estimated to be 1,966 CFU using Probit analysis. Following the LD50 determination, pathology was evaluated by exposing mice to a target LD99 (42,890 CFU). Mice were euthanized at 12, 24, 36, 48, 60, and 72 h postexposure. At each time point, samples were collected for clinical pathology, detection of bacteria in blood and tissues, and pathology evaluations. A general increase in incidence and severity of microscopic findings was observed in the lung, lymph nodes, spleen, and liver from 36 to 72 h postchallenge. Similarly, the incidence and severity of pneumonia increased throughout the study; however, some mice died in the absence of pneumonia, suggesting that disease progression does not require the development of pneumonia. Disease pathology in the Swiss Webster mouse is similar to that observed in humans, demonstrating the utility of this pneumonic plague model that can be used by researchers investigating plague countermeasures.
Yersinia pestis causes pneumonic plague, an exceptionally virulent disease for which we lack a safe and effective vaccine. Antibodies specific for the Y. pestis F1 and LcrV proteins can protect mice against pulmonary Y. pestis infection. We demonstrate that neutralizing tumor necrosis factor-alpha (TNFα) and gamma-interferon (IFNγ) abrogates this protection at sub-optimal levels of F1- or LcrV-specific antibody, but not at optimal levels. Moreover, we demonstrate that endogenous TNFα and IFNγ confer measurable protection in the complete absence of protective antibodies. These findings indicate that antibodies and cytokines independently protect against pneumonic plague and suggest that surrogate assays for plague vaccine efficacy should consider both the level of vaccine-induced antibody and the capacity of vaccine recipients to produce TNFα and IFNγ upon exposure to Y. pestis.
Yersinia pestis; vaccine; antibody; cytokine
The potential use of Yersinia pestis as a bioterror agent is a great concern. Development of a stable powder vaccine against Y. pestis and administration of the vaccine by minimally invasive methods could provide an alternative to the traditional liquid formulation and intramuscular injection. We evaluated a spray-freeze-dried powder vaccine containing a recombinant F1-V fusion protein of Y. pestis for vaccination against plaque in a mouse model. Mice were immunized with reconstituted spray-freeze-dried F1-V powder via intramuscular injection, microneedle-based intradermal delivery, or noninvasive intranasal administration. By intramuscular injection, the reconstituted powder induced serum antibody responses and provided protection against lethal subcutaneous challenge with 1,000 50% lethal doses of Y. pestis at levels equivalent to those elicited by unprocessed liquid formulations (70 to 90% protection). The feasibility of intradermal and intranasal delivery of reconstituted powder F1-V vaccine was also demonstrated. Overall, microneedle-based intradermal delivery was shown to be similar in efficacy to intramuscular injection, while intranasal administration required an extra dose of vaccine to achieve similar protection. In addition, the results suggest that seroconversion against F1 may be a better predictor of protection against Y. pestis challenge than seroconversion against either F1-V or V. In summary, we demonstrate the preclinical feasibility of using a reconstituted powder F1-V formulation and microneedle-based intradermal delivery to provide protective immunity against plague in a mouse model. Intranasal delivery, while feasible, was less effective than injection in this study. The potential use of these alternative delivery methods and a powder vaccine formulation may result in substantial health and economic benefits.
Yersinia pestis, the bacterium that causes plague, is a potential agent of biowarfare and bioterrorism. The aminoglycoside antibiotic streptomycin is the gold standard for treatment. However, this recommendation is based on scant animal and clinical data. We used an in vitro pharmacodynamic infection model to compare the efficacies of 10-day regimens of streptomycin versus the fluoroquinolone antibiotic levofloxacin for the treatment of Y. pestis infection and to evaluate for emergence of resistance. The human serum concentration-time profiles for standard clinical regimens of 1 g of streptomycin given every 12 h and 500 mg of levofloxacin given every 24 h were simulated. The growth fitness of drug-resistant mutants was examined in neutropenic and immunocompetent mouse thigh infection models. In the in vitro infection system, untreated bacteria grew from 107 to 1010 CFU/ml. Streptomycin therapy caused a 105 CFU/ml reduction in the number of bacteria over 24 h, followed by regrowth with streptomycin-resistant mutants. Levofloxacin resulted in a 107 CFU/ml reduction in the number of bacteria within 12 h, ultimately sterilizing the culture without resistance selection. In both the normal and neutropenic mouse infection models, streptomycin-resistant and wild-type strains were equally fit. However, 90% of levofloxacin-resistant isolates, cultured from the control in vitro infection arm, did not proliferate in the mouse models. Thus, the fluoroquinolone antibiotic levofloxacin was superior to streptomycin in our in vitro infection model. The majority of levofloxacin-resistant mutants were less fit than streptomycin-resistant and wild-type Y. pestis.
Yersinia pestis, the causative agent of plague, secretes LcrV (low-calcium-response V or V antigen) during infection. LcrV triggers the release of interleukin 10 (IL-10) by host immune cells and suppresses proinflammatory cytokines such as tumor necrosis factor alpha and gamma interferon as well as innate defense mechanisms required to combat the pathogenesis of plague. Although immunization of animals with LcrV elicits protective immunity, the associated suppression of host defense mechanisms may preclude the use of LcrV as a human vaccine. Here we show that short deletions within LcrV can reduce its immune modulatory properties. An LcrV variant lacking amino acid residues 271 to 300 (rV10) elicited immune responses that protected mice against a lethal challenge with Y. pestis. Compared to full-length LcrV, rV10 displayed a reduced ability to release IL-10 from mouse and human macrophages. Furthermore, the lipopolysaccharide-stimulated release of proinflammatory cytokines by human or mouse macrophages was inhibited by full-length LcrV but not by the rV10 variant. Thus, it appears that LcrV variants with reduced immune modulatory properties could be used as a human vaccine to generate protective immunity against plague.
Septic bacterial pneumonias are a major cause of death worldwide. Several of the highest priority bioterror concerns, including anthrax, tularemia and plague, are caused by bacteria that acutely infect the lung. Bacterial resistance to multiple antibiotics is increasingly common. Although vaccines may be our best defense against antibiotic-resistant bacteria, there has been little progress in the development of safe and effective vaccines for pulmonary bacterial pathogens. The gram-negative bacterium Yersinia pestis causes pneumonic plague, an acutely lethal septic pneumonia. Historic pandemics of plague caused millions of deaths, and the plague bacilli’s potential for weaponization sustains an ongoing quest for effective countermeasures. Subunit vaccines have failed, thus far, to fully protect non-human primates. In mice, they induce the production of antibodies that act in concert with type 1 cytokines to deliver high-level protection, however, the Y. pestis antigens recognized by cytokine-producing T cells have yet to be defined. Here, we report that Y. pestis YopE is a dominant antigen recognized by CD8 T cells in C57BL/6 mice. After vaccinating with live attenuated Y. pestis and challenging intranasally with virulent plague, nearly 20% of pulmonary CD8 T cells recognize this single, highly conserved antigen. Moreover, immunizing mice with a single peptide, YopE69-77, suffices to confer significant protection from lethal pulmonary challenge. These findings suggest YopE could be a valuable addition to subunit plague vaccines and provide a new animal model in which sensitive, pathogen-specific assays can be used to study CD8 T cell-mediated defense against acutely lethal bacterial infections of the lung.
An attenuated Salmonella typhimurium strain which expressed the F1 capsular antigen of Yersinia pestis was constructed by transformation of S. typhimurium SL3261 with plasmid pFGAL2a, a derivative of pUC18 which contained the caf1 gene without the leader sequence. The recombinant was used to vaccinate mice intragastrically and intravenously. The immunity induced was able to protect mice against challenge with a virulent strain of plague. Protection correlated with the induction of high titers of immunoglobulin G in serum samples and a specific T-cell response.
Burkholderia pseudomallei is a Category B select agent and the cause of melioidosis. Research funding for vaccine development has largely considered protection within the biothreat context, but the resulting vaccines could be applicable to populations who are at risk of naturally acquired melioidosis. Here, we discuss target populations for vaccination, consider the cost-benefit of different vaccination strategies and review potential vaccine candidates.
Methods and Findings
Melioidosis is highly endemic in Thailand and northern Australia, where a biodefense vaccine might be adopted for public health purposes. A cost-effectiveness analysis model was developed, which showed that a vaccine could be a cost-effective intervention in Thailand, particularly if used in high-risk populations such as diabetics. Cost-effectiveness was observed in a model in which only partial immunity was assumed. The review systematically summarized all melioidosis vaccine candidates and studies in animal models that had evaluated their protectiveness. Possible candidates included live attenuated, whole cell killed, sub-unit, plasmid DNA and dendritic cell vaccines. Live attenuated vaccines were not considered favorably because of possible reversion to virulence and hypothetical risk of latent infection, while the other candidates need further development and evaluation. Melioidosis is acquired by skin inoculation, inhalation and ingestion, but routes of animal inoculation in most published studies to date do not reflect all of this. We found a lack of studies using diabetic models, which will be central to any evaluation of a melioidosis vaccine for natural infection since diabetes is the most important risk factor.
Vaccines could represent one strand of a public health initiative to reduce the global incidence of melioidosis.
The designation of Burkholderia pseudomallei as a category B select agent has resulted in considerable research funding to develop a protective vaccine. This bacterium also causes a naturally occurring disease (melioidosis), an important cause of death in many countries including Thailand and Australia. In this study, we explored whether a vaccine could be used to provide protection from melioidosis. An economic evaluation based on its use in Thailand indicated that a vaccine could be a cost-effective intervention if used in high-risk populations such as diabetics and those with chronic kidney or lung disease. A literature search of vaccine studies in animal models identified the current candidates, but noted that models failed to take account of the common routes of infection in natural melioidosis and major risk factors for infection, primarily diabetes. This review highlights important areas for future research if biodefence-driven vaccines are to play a role in reducing the global incidence of melioidosis.
Burkholderia pseudomallei is the causative agent of melioidosis, a rare disease of biodefense concern with high mortality and extreme difficulty in treatment. No human vaccines are available that protect against B. pseudomallei infection, and with the current limitations of antibiotic treatment, the development of new preventative and therapeutic interventions is crucial. Although clinical trials could be used to test the efficacy of new medical countermeasures (MCMs), the high mortality rates associated with melioidosis raises significant ethical issues concerning treating individuals with new compounds with unknown efficacies. The US Food and Drug Administration (FDA) has formulated a set of guidelines for the licensure of new MCMs to treat diseases in which it would be unethical to test the efficacy of these drugs in humans. The FDA “Animal Rule” 21 CFR 314 calls for consistent, well-characterized B. pseudomallei strains to be used as challenge material in animal models. In order to facilitate the efficacy testing of new MCMs for melioidosis using animal models, we intend to develop a well-characterized panel of strains for use. This panel will comprise of strains that were isolated from human cases, have a low passage history, are virulent in animal models, and are well-characterized phenotypically and genotypically. We have reviewed published and unpublished data on various B. pseudomallei strains to establish an objective method for selecting the strains to be included in the panel of B. pseudomallei strains with attention to five categories: animal infection models, genetic characterization, clinical and passage history, and availability of the strain to the research community. We identified 109 strains with data in at least one of the five categories, scored each strain based on the gathered data and identified six strains as candidate for a B. pseudomallei strain panel.
pseudomallei; aerosol; biodefense; animal models; virulence; genome; sequencing; passage history
Combination vaccines reduce the total number of injections required for each component administered separately and generally provide the same level of disease protection. Yet, physical, chemical, and biological interactions between vaccine components are often detrimental to vaccine safety or efficacy.
As a possible alternative to combination vaccines, we used specially designed microneedles to inject rhesus macaques with four separate recombinant protein vaccines for anthrax, botulism, plague and staphylococcal toxic shock next to each other just below the surface of the skin, thus avoiding potentially incompatible vaccine mixtures.
The intradermally-administered vaccines retained potent antibody responses and were well- tolerated by rhesus macaques. Based on tracking of the adjuvant, the vaccines were transported from the dermis to draining lymph nodes by antigen-presenting cells. Vaccinated primates were completely protected from an otherwise lethal aerosol challenge by Bacillus anthracis spores, botulinum neurotoxin A, or staphylococcal enterotoxin B.
Our results demonstrated that the physical separation of vaccines both in the syringe and at the site of administration did not adversely affect the biological activity of each component.
The vaccination method we describe may be scalable to include a greater number of antigens, while avoiding the physical and chemical incompatibilities encountered by combining multiple vaccines together in one product.
Identification of an immune response to vaccination that reliably predicts protection from clinically significant infection, i.e. an immunological surrogate endpoint, is a primary goal of vaccine research. Using this problem of evaluating an immunological surrogate as an illustration, we describe a hierarchy of three criteria for a valid surrogate endpoint and statistical analysis frameworks for evaluating them. Based on a placebo-controlled vaccine efficacy trial, the first level entails assessing the correlation of an immune response with a study endpoint in the study groups, and the second level entails evaluating an immune response as a surrogate for the study endpoint that can be used for predicting vaccine efficacy for a setting similar to that of the vaccine trial. We show that baseline covariates, innovative study design, and a potential outcomes formulation can be helpful for this assessment. The third level entails validation of a surrogate endpoint via meta-analysis, where the goal is to evaluate how well the immune response can be used to predict vaccine efficacy for new settings (building bridges). A simulated vaccine trial and two example vaccine trials are presented, one supporting that certain anti-influenza antibody levels are an excellent surrogate for influenza illness and another supporting that certain anti-HIV antibody levels are not useful as a surrogate for HIV infection.
clinical trial; counterfactual; immune correlate; meta-analysis; potential outcomes; principal surrogate; statistical surrogate
The chemokine, lymphotactin (LTN), was tested as a molecular adjuvant using bicistronic DNA vaccines encoding the protective Yersinia capsular (F1) antigen and virulence antigen (V-Ag) as a F1-V fusion protein. The LTN-encoding F1-V or V-Ag vaccines were given by the intranasal (i.n.) or intramuscular (i.m.) routes, and although serum IgG and mucosal IgA antibodies (Abs) were induced, F1-Ag boosts were required for robust anti-F1-Ag Abs. Optimal efficacy against pneumonic plague was obtained in mice i.m.-, not i.n.-immunized with these DNA vaccines. These vaccines stimulated elevated Ag-specific Ab-forming cells and mixed Th cell responses, with Th17 cells markedly enhanced by i.m. immunization. These results show that LTN can be used as a molecular adjuvant to enhance protective immunity against plague.
mucosal; IgA; vaccines; pneumonic plague; lymphotactin
Pneumonic plague remains problematic in endemic areas, and because it can be readily transmitted and has high mortality, the development of efficacious vaccines is warranted. To test whether stimulation of cell-mediated immunity with IL-12 will improve protective immunity against plague, we constructed two IL-12 DNA vaccines using a bicistronic plasmid encoding the protective plague epitopes, capsular (F1) antigen and virulence antigen (V-Ag) as F1-V fusion protein and V-Ag only, respectively. When applied intramuscularly, antibody responses to F1- and V-Ag were detectable beginning at week 6 after three weekly doses, and F1-Ag protein boosts were required to induce elevated Ab responses. These Ab responses were supported by mixed Th cell responses, and the IL-12/V-Ag DNA vaccine showed greater cell-mediated immune bias than IL-12/F1-V DNA vaccine. Following pneumonic challenge, both IL-12 DNA vaccines showed similar efficacy despite differences in Th cells simulated. These results show that IL-12 can be used as a molecular adjuvant to enhance protective immunity against pneumonic plague.
IL-12; Pneumonic plague; DNA vaccine
In our previous study, complete protection was observed in Chinese-origin rhesus macaques immunized with SV1 (20 µg F1 and 10 µg rV270) and SV2 (200 µg F1 and 100 µg rV270) subunit vaccines and with EV76 live attenuated vaccine against subcutaneous challenge with 6×106 CFU of Y. pestis. In the present study, we investigated whether the vaccines can effectively protect immunized animals from any pathologic changes using histological and immunohistochemical techniques. In addition, the glomerular basement membranes (GBMs) of the immunized animals and control animals were checked by electron microscopy. The results show no signs of histopathological lesions in the lungs, livers, kidneys, lymph nodes, spleens and hearts of the immunized animals at Day 14 after the challenge, whereas pathological alterations were seen in the corresponding tissues of the control animals. Giemsa staining, ultrastructural examination, and immunohistochemical staining revealed bacteria in some of the organs of the control animals, whereas no bacterium was observed among the immunized animals. Ultrastructural observation revealed that no glomerular immune deposits on the GBM. These observations suggest that the vaccines can effectively protect animals from any pathologic changes and eliminate Y. pestis from the immunized animals. The control animals died from multi-organ lesions specifically caused by the Y. pestis infection. We also found that subcutaneous infection of animals with Y. pestis results in bubonic plague, followed by pneumonic and septicemic plagues. The histopathologic features of plague in rhesus macaques closely resemble those of rodent and human plagues. Thus, Chinese-origin rhesus macaques serve as useful models in studying Y. pestis pathogenesis, host response and the efficacy of new medical countermeasures against plague.
In contrast to Yersinia pestis LcrV, the recombinant V10 (rV10) variant (lacking residues 271 to 300) does not suppress the release of proinflammatory cytokines by immune cells. Immunization with rV10 generates robust antibody responses that protect mice against bubonic plague and pneumonic plague, suggesting that rV10 may serve as an improved plague vaccine.