Model For Vaccine Development Research Articles

Discovery and development of COVID-19 vaccine from laboratory to clinic.

The world has recently experienced one of the biggest and most severe public health disasters with severe acute respiratory syndrome coronavirus (SARS-CoV-2). SARS-CoV-2 is responsible for the coronavirus disease of 2019 (COVID-19) which is one of the most widespread and powerful infections affecting human lungs. Current figures show that the epidemic had reached 216 nations, where it had killed about 6,438,926 individuals and infected 590,405,710. WHO proclaimed the outbreak of the Ebola virus disease (EVD), in 2014 that killed hundreds of people in West Africa. The development of vaccines for SARS-CoV-2 becomes more difficult due to the viral mutation in its non-structural proteins (NSPs) especially NSP2 and NSP3, S protein, and RNA-dependent RNA polymerase (RdRp). Continuous monitoring of SARS-CoV-2, dynamics of the genomic sequence, and spike protein mutations are very important for the successful development of vaccines with good efficacy. Hence, the vaccine development for SARS-CoV-2 faces specific challenges starting from viral mutation. The requirement of long-term immunity development, safety, efficacy, stability, vaccine allocation, distribution, and finally, its cost is discussed in detail. Currently, 169 vaccines are in the clinical development stage, while 198 vaccines are in the preclinical development stage. The majority of these vaccines belong to the Ps-Protein subunit type which has 54, and the minor BacAg-SPV (Bacterial antigen-spore expression vector) type, at least 1 vaccination. The use of computational methods and models for vaccine development has revolutionized the traditional methods of vaccine development. Further, this updated review highlights the upcoming vaccine development strategies in response to the current pandemic and post-pandemic era, in the field of vaccine development.

Tetravalent SARS-CoV-2 S1 subunit protein vaccination elicits robust humoral and cellular immune responses in SIV-infected rhesus macaque controllers.

The study provides important insights into the immunogenicity and efficacy of a tetravalent protein subunit vaccine candidate against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The vaccine induced both humoral and cellular immune responses in nonhuman primates with controlled SIVagm infection and was able to generate Omicron variant-specific antibodies without specifically vaccinating with Omicron. These findings suggest that the tetravalent composition of the vaccine candidate could provide broad protection against multiple SARS-CoV-2 variants while minimizing the risk of immune escape and the emergence of new variants. Additionally, the use of rhesus macaques with controlled SIVsab infection may better represent vaccine immunogenicity in humans with chronic viral diseases, highlighting the importance of preclinical animal models in vaccine development. Overall, the study provides valuable information for the development and implementation of coronavirus disease 2019 vaccines, particularly for achieving global vaccine equity and addressing emerging variants.

Genomic evolution of the SARS-CoV-2 Variants of Concern: COVID-19 pandemic waves in India.

SARS-CoV-2 has mutated rapidly since its first case report in Wuhan, China, leading to the emergence of an indefinite number of variants. India has witnessed three waves of the COVID-19 pandemic. The country saw its first wave of SARS-CoV-2 illness from late January 2020 to February 2021. With a peak surge of cases in mid-September 2020, India recorded more than 11 million cases and a death toll of more than 0.165 million at this time. India faced a brutal second wave driven by the emergence of highly infectious SARS-CoV-2 variants B.1.617.2 (Delta variant) and the third wave with the leading cause of BA.2 (Omicron variant), which has led to an unprecedented rise in COVID-19 cases in the country. On September 14, 2022, India recorded a cumulative 44.51 million cases of COVID-19 with more than 0.528 million deaths. The discovery of common circulating mutants is facilitated by genome sequencing. The changes in the Spike surface glycoprotein recombinant binding domains served as the critical alterations, resulting in enhanced infectivity and transmissibility, with severe clinical effects. Further, the predominant mutation in the SARS-CoV-2 spike protein; the D614G strains served as a model for vaccine development. The mutation of the Wuhan strain to the Variant of Concern led to a significant increase in SARS-CoV-2 infections. In addition, there was a shift in the age group affected by SARS-CoV-2 variant infection. The current review summarized the COVID-19 pandemic's Variant of Concern and the advent of SARS-CoV-2 in India.

Duration of peripheral blood cellular immune responses induced in dogs in response to a modified live attenuated vaccine for Ehrlichia chaffeensis

Abstract Ehrlichia chaffeensis (ECH) is an obligate intracellular gram-negative bacterium and a frequent cause of severe and fatal tick-borne infection in people in North America. The canine is a common incidental host and a useful model for vaccine development and assessing immune responses to ECH infection. We recently established mutagenesis methods for ECH and demonstrated that immunization of dogs with an ECH mutant with a functional disruption in the ECH_0660 gene protected the vertebrate host from both tick-transmitted and intravenous wildtype ECH challenge when given 4 weeks after immunization. In the current study, we evaluated the duration of immunity offered by the ECH_0660 gene mutant live attenuated vaccine (MLAV) against wildtype infection challenge. Groups of 12 dogs were immunized with the ECH MLAV and then challenged 2, 4, 8 and 12 months later via tick-transmitted or blood-transmitted infection with wildtype ECH. Animals were monitored for 4 weeks after challenge and then sacrificed for evaluation of tissue pathology and bacterial burden. Immunization with the MLAV induced ECH-specific T cell responses, detectable by ELISPOT for IFNγ; and differentiation of IFNγ- and/or TNFα-producing ECH-specific T effector memory CD4 T cells (CD45RAneg CD45RO+ CD62Lneg). Antigen-specific recall responses were detectable in the peripheral blood of most (27/32) vaccinated dogs for 4 months after immunization, but only in 5/24 of the dogs after 8 months. This result suggests that a single MLAV vaccination induces cellular immune responses and differentiation of memory helper T cells. Future work will investigate the correlations between cellular immunity, humoral responses and bacterial load. Supported by grants from NIH (R01 AI152418)

Animal experiments show impact of vaccination on reduction of SARS-CoV-2 virus circulation: A model for vaccine development?

BackgroundIn the pre-clinical phase, SARS-CoV-2 vaccines were tested in animal models, including exposure trials, to investigate protection against SARS-CoV-2. These studies paved the way for clinical development. The objective of our review was to provide an overview of published animal exposure results, focussing on the capacity of vaccines to reduce/prevent viral shedding. MethodUsing Medline, we retrieved eighteen papers on eight different vaccine platforms in four animal models. Data were extracted on presence/absence of viral RNA in nose, throat, or lungs, and neutralizing antibody levels in the blood. ResultsAll vaccines showed a tendency of reduced viral load after exposure. Particularly nasal swab results are likely to give an indication about the impact on virus excretion in the environment. Similarly, the reduction or prevention of viral replication in the bronchoalveolar environment might be related with disease prevention, explaining the high efficacy in clinical trials. DiscussionAlthough it remains difficult to compare the results directly, the potential for a strong reduction of transmission was shown, indicating that the animal models predicted what is observed in the field after large scale human vaccination. This merits further attention for standardization of exposure experiments, with the intention to speed up future vaccine development.

Establishment of a nonhuman primate model for development of vaccines and anti-viral drugs against COVID-19

Establishment of a nonhuman primate model for development of vaccines and anti-viral drugs against COVID-19

Passive immunisation of convalescent human anti-Zika plasma protects against challenge with New World Zika virus in cynomolgus macaques

Zika virus (ZIKV) causes neurological complications in susceptible individuals, highlighted in the recent South American epidemic. Natural ZIKV infection elicits host responses capable of preventing subsequent re-infection, raising expectations for effective vaccination. Defining protective immune correlates will inform viral intervention strategies, particularly vaccine development. Non-human primate (NHP) species are susceptible to ZIKV and represent models for vaccine development. The protective efficacy of a human anti-ZIKV convalescent plasma pool (16/320-14) developed as a candidate reference material for a WHO International Standard was evaluated in macaques. Convalescent plasma administered to four cynomolgus macaques (Macaca fascicularis) intra-peritoneally 24 hrs prior to sub-cutaneous challenge with 103 pfu ZIKVPRVABC59 protected against detectable infection, with absence of detectable ZIKV RNA in blood and lymphoid tissues. Passively immunised anti-ZIKV immunoglobulin administered prior to time of challenge remained present only at very low levels 42 days post-challenge. Absence of de novo antibody responses in passively immunised macaques indicate sterilising immunity compared with naïve challenge controls that exhibited active ZIKV-specific IgM and IgG responses post-challenge. Demonstration that the presence of convalescent anti-ZIKV at levels of 400 IU/mL neutralising antibody protects against virus challenge provides a scientific framework for development of anti-ZIKV vaccines and facilitates regulatory approval.

Commentary on two reports on animal models of COVID-19.

Commentary on two reports on animal models of COVID-19.

An adjuvanted adenovirus 5-based vaccine elicits neutralizing antibodies and protects mice against chikungunya virus-induced footpad swelling

Over the past decade, chikungunya virus (CHIKV) has emerged as a major cause of mosquito-borne disease with transmission reported in over 100 countries worldwide. Although several strategies have been pursued for the development of a CHIKV vaccine, none has been approved yet. In this study, we describe the development of several vaccine vectors that express the structural proteins of the La Réunion CHIKV strain LR2006-OPY1. Protection from virus-induced pathologic changes was observed in vaccinated C57BL/6 mice, an important model for CHIKV vaccine development because of their ability to recapitulate several signs shown in infected humans. This study uniquely demonstrates the capacity of a mucosally-administered adenovirus vaccine to induce serum antibody responses and confer protective efficacy in a pre-clinical model. Our data provide further evidence in support of the clinical development of this oral Ad-CHIKV vaccine strategy in populations at high risk of contracting the disease.

Recent developments for Staphylococcus aureus vaccines: clinical and basic science challenges.

Bacterial vaccines have made dramatic impacts upon morbidity and mortality caused by a number of common pathogens, but a vaccine to prevent Staphylococcus aureus infections has proven to be illusive. With successful bacterial vaccines, the organisms are all part of the transient flora, whereas, S. aureus is part of the normal human flora. This means that S. aureus has had a prolonged time to adapt to the host milieu and its defences. The failure of several staphylococcal antigens to protect humans from infection in vaccine clinical trials using active or passive immunisation has stimulated a re-examination of the fundamental assumptions about staphylococcal immunity in humans vs. animals, especially rodents. This has spurred an active debate about the appropriate models for vaccine development and an examination of our current understanding of the protective immunity in humans. A major factor in the development of previous bacterial vaccines was a biomarker that predicted human protection, e.g., antibodies to tetanus toxoid or to pneumococcal polysaccharide. While antibodies against a number of staphylococcal antigens have proven to be an excellent biomarker for protection in rodents, these have not been translated to human infections. Thus, while much work remains, there is a growing consensus that T-cell immunity plays an important role in protecting humans. Moreover, the presence of anti-staphylococcal toxin antibodies correlates with reduced disease severity in humans. The most important recent advances concerning potential biomarkers, and the role of pre-existing immune status of vaccines in vaccine-associated mortality are considered in this review.

Delivery of Human EV71 Receptors by Adeno-Associated Virus Increases EV71 Infection-Induced Local Inflammation in Adult Mice

Enterovirus71 (EV71) is now recognized as an emerging neurotropic virus in Asia and one major causative agent of hand-foot-mouth diseases (HFMD). However potential animal models for vaccine development are limited to young mice. In this study, we used an adeno-associated virus (AAV) vector to introduce the human EV71 receptors P-selectin glycoprotein ligand-1 (hPSGL1) or a scavenger receptor class-B member-2 (hSCARB2) into adult ICR mice to change their susceptibility to EV71 infection. Mice were administered AAV-hSCARB2 or AAV-hPSGL1 through intravenous and oral routes. After three weeks, expression of human SCARB2 and PSGL1 was detected in various organs. After infection with EV71, we found that the EV71 viral load in AAV-hSCARB2- or AAV-hPSGL1-transduced mice was higher than that of the control mice in both the brain and intestines. The presence of EV71 viral particles in tissues was confirmed using immunohistochemistry analysis. Moreover, inflammatory cytokines were induced in the brain and intestines of AAV-hSCARB2- or AAV-hPSGL1-transduced mice after EV71 infection but not in wild-type mice. However, neurological disease was not observed in these animals. Taken together, we successfully infected adult mice with live EV71 and induced local inflammation using an AAV delivery system.

Characterization and allelic polymorphisms of rhesus macaque (Macaca mulatta) IgG Fc receptor genes

Macaque models are invaluable for AIDS research. Indeed, initial development of HIV-1 vaccines relies heavily on simian immunodeficiency virus-infected rhesus macaques. Neutralizing antibodies, a major component of anti-HIV protective responses, ultimately interact with Fc receptors on phagocytic and natural killer cells to eliminate the pathogen. Despite the major role that Fc receptors play in protective responses, there is very limited information available on these molecules in rhesus macaques. Therefore, in this study, rhesus macaque CD32 (FcγRII) and CD64 (FcγRI) homologues were genetically characterized. In addition, presence of CD16 (FcγRIII), CD32, and CD64 allelic polymorphisms were determined in a group of nine animals. Results from this study show that the predicted structures of macaque CD32 and CD64 are highly similar to their human counterparts. Macaque and human CD32 and CD64 extracellular domains are 88-90% and 94-95% homologous, respectively. Although all cysteines are conserved between the two species, macaque CD32 exhibits two additional N-linked glycosylation sites, whereas CD64 lacks three of them when compared to humans. Five CD32, three CD64, and three CD16 distinct allelic sequences were indentified in the nine animals examined, indicating a relatively high level of polymorphism in macaque Fcγ receptors. Together, these results validate rhesus macaques as models for vaccine development and antibody responses, while at the same time, underscoring the need to take into account the high degree of genetic heterogeneity present in this species when designing experimental protocols.

Abstract 5631: Nitric oxide-based immune response modifications: A novel paradigm in tumor vaccine development against melanoma

Abstract Although of cardinal importance, the specific role of nitric oxide (NO) in the immune response to cancer remains elusive. NO is generally considered to act as a terminal effector molecule that will promote direct cell death due to its cytotoxic and/or cytostatic properties. Conversely, NO has been also found to be associated with a cytoprotective effect due to its ability to interfere with the apoptotic machinery. We have previously shown that NO is capable to immuno-sensitized tumor cells by regulating the expression of apoptosis-related genes and modifying the structure of many protein targets. Thus, we hypothesize that NO-based modification of an autologous tumor vaccine preparation may improve the immunological priming against tumor cells and potentiate the specific anti-tumor immune response. In order to test this hypothesis we used the well-characterized B16-C57BL/6 syngeneic mouse model of melanoma. B16-F0 melanoma cells were cultured in the presence or absence of a non-cytotoxic concentration of a slow rate release NO donor for 18 hours, and later homogenized into a crude lysate. These preparations were used to vaccinate mice previously challenged with B16-F0 melanoma cells on the contralateral dorsal flank one week before, in order to frame a therapeutic paradigm of treatment in cancer. Additional doses of vaccine were applied every 7 days for a period of 3 weeks and tumor volume was determined every 3 days. Our results demonstrate that the NO-modified autologous cancer vaccine decreases the rate of tumor growth by approximately 85% (p <0.001) by day 25 when compared with the control groups. In addition, this vaccination scheme increases the survival rate by approximately 60% (p <0.001) of the experimental group when compared with controls. NO can exert a myriad of modifications at the genetic, epigenetic and post-translational level in the tumor cells promoting a robust and effective therapeutic immune response against cancer cells. Currently, we are defining the molecular and immunological mechanisms responsible for the significant increase in survival and anti-tumor immune response triggered by the NO-mediated modifications of the melanoma tumor cells. Our results suggest a new role of NO as an immune response modifier that can be exploited in cancer immunotherapy and serves as a model for vaccine development. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5631.

Maintenance or Emergence of Chronic Phase Secondary Cytotoxic T Lymphocyte Responses after Loss of Acute Phase Immunodominant Responses Does Not Protect SIV-Infected Rhesus Macaques from Disease Progression

The simian immunodeficiency virus- (SIV-) infected rhesus macaque is the preferred animal model for vaccine development, but the correlates of protection in this model are not completely understood. In this paper, we document the cytotoxic T lymphocyte (CTL) response to SIV and its effects on viral evolution in an effort to identify events associated with disease progression regardless of MHC allele expression. We observed the evolution of epitopes targeted by CTLs in a group of macaques that included long-term nonprogressing (LTNP), slowly progressing (SP), normally progressing (NP), and rapidly progressing (RP) animals. Collectively, our data (1) identify novel CTL epitopes from an SP animal that are not restricted by known protective alleles, (2) illustrate that, in this small study, RP and NP animals accrue more mutations in CTL epitopes than in SP or LTNP macaques, and (3) demonstrate that the loss of CTL responses to immunodominant epitopes is associated with viral replication increases, which are not controlled by secondary CTL responses. These findings provide further evidence for the critical role of the primary cell-mediated immune responses in the control of retroviral infections.

Model for product development of vaccines against neglected tropical diseases: a vaccine against human hookworm

This article provides an overview of the advances in product development and technology transfer of the vaccine against human hookworm, with particular emphasis on the lessons learned and the challenges of developing a vaccine in the nonprofit sector. The comprehensive approach to vaccine development established by the Human Hookworm Vaccine Initiative (HHVI) identifies key operational and technical aspects that are essential for a successful partnership with a developing country vaccine manufacturer. This article also highlights the importance of a global access roadmap to guide the vaccine development program. The advancement of new products for the control of neglected tropical diseases portends great challenges for global access, including aspects related to vaccine design, product development and manufacture, vaccine introduction and distribution, financing, knowledge dissemination and intellectual property management. With only three vaccines for neglected tropical diseases in clinical trials – hookworm, leishmaniasis and schistosomiasis – we are at the nascent stages of developing vaccines for neglected populations. Product development public–private partnerships, such as the HHVI, continue to show great promise on this front and will eventually provide significant control tools for achieving millennium development goals related to poverty reduction, as well as child and maternal health.

Cattle as a model for development of vaccines against human tuberculosis

The identification of tuberculosis vaccines and vaccination strategies, which in small animal models appear to be more effective than BCG, offer some exciting possibilities for control of human tuberculosis in the future. However, some major problems remain including selecting which vaccines should go into human trials and the length of time it will take for testing these vaccines in humans. The cattle model is well suited for the secondary screening of tuberculosis vaccines as there is a strong similarity between the disease in cattle and humans and outbred animals are used. Moreover, there are many similarities in the results from field trials of BCG in both cattle and humans, with BCG often failing to protect when the trials are extended over a number of years. In addition, calves, like human infants, are immunocompetent at birth. Recent studies in calves have shown that BCG vaccination of calves within hours of birth is highly effective in protecting animals against bovine tuberculosis, but BCG revaccination at 6 weeks of age is contraindicated. Prime boost vaccination strategies using BCG and DNA vaccines have provided evidence that these combinations may give better protection in calves than either vaccine alone. Based on antigens whose genes are absent from the BCG genome, advances have also been made to develop diagnostic reagents distinguishing infected and vaccinated animals (differential diagnosis). The cattle model has been particularly useful in prioritizing such antigens for testing in humans. Finally, there is an urgent need to identify an immunological correlate of protection against tuberculosis. The cattle model can be particularly helpful in this area as it is relatively easy to collect large volumes of blood from calves at intervals following vaccination and challenge, and a large number of immunological reagents are now available for cattle.

Human Autologous Tumor-Specific T-Cell Responses Induced by Liposomal Delivery of a Lymphoma Antigen

The idiotype (Id) of the immunoglobulin on a given B-cell malignancy is a clonal marker that can serve as a tumor-specific antigen. We developed a novel vaccine formulation by incorporating Id protein with liposomal lymphokine that was more potent than a prototype, carrier-conjugated Id protein vaccine in preclinical studies. In the present study, we evaluated the safety and immunogenicity of this vaccine in follicular lymphoma patients. Ten patients with advanced-stage follicular lymphoma were treated with five doses of this second generation vaccine after chemotherapy-induced clinical remission. All patients were evaluated for cellular and humoral immune responses. Autologous tumor and Id-specific type I cytokine responses were induced by vaccination in 10 and 9 patients, respectively. Antitumor immune responses were mediated by both CD4+ and CD8+ T cells, were human lymphocyte antigen class I and II associated, and persisted 18 months beyond the completion of vaccination. Specific anti-Id antibody responses were detected in four patients. After a median follow-up of 50 months, 6 of the 10 patients remain in continuous first complete remission. This first clinical report of a liposomal cancer vaccine demonstrates that liposomal delivery is safe, induces sustained tumor-specific CD4+ and CD8+ T-cell responses in lymphoma patients, and may serve as a model for vaccine development against other human cancers and infectious pathogens.

Human Autologous Tumor-Specific T-Cell Responses Induced by Liposome Encapsulated Lymphoma Membrane Proteins.

The unique antigenic determinants (Idiotype [Id]) of the immunoglobulin expressed on a given B cell malignancy can serve as a tumor-specific antigen for active immunotherapy. We have previously demonstrated that Id vaccines in follicular lymphoma (FL) patients administered in the minimal residual disease (MRD) state are immunogenic and are associated with induction of complete molecular remissions and long-term disease-free survival (Nature Med 5:1171–1177, 1999). This hybridoma-derived vaccine is now being tested in a pivotal Phase III clinical trial. However, the production of Id protein by hybridoma technology for such vaccine formulation is an expensive and laborious process requiring an average of 3 to 6 months to manufacture the vaccine for each patient. To overcome this difficulty, we developed a novel vaccine formulation where we directly extracted the membrane proteins from lymph node biopsy-derived tumor cells and incorporated them into liposomes along with IL-2. Testing in preclinical studies showed this formulation to be as potent as our prototype hybridoma-derived Id protein vaccine. In the present study, 11 previously untreated and/or relapsed FL patients received 5 injections of this novel vaccine formulation subcutaneously and/or intratumorally at approximately monthly intervals. The vaccine was well tolerated and induced only minor local reactions at the sites of injection. T cell responses were evaluated by cytokine induction and IFNg ELISPOT against autologous tumor. Post-vaccine, but not pre-vaccine, peripheral blood mononuclear cells (PBMC) from 6 out of 10 patients that were assessed, recognized autologous tumor cells, as demonstrated by TNFa, GM-CSF and/or IFNg production. Significant production of cytokines was observed only in response to autologous tumor cells, but not normal B cells. The precursor frequency of tumor-reactive T cells was significantly increased in postvaccine PBMC (range 19–115 IFNg spots/100,000 PBMC), compared with prevaccine PBMC (range 2–7 IFNg spots/100,000 PBMC). Anti-MHC Class I and Class II antibodies inhibited cytokine production suggesting that both CD4+ and CD8+ T cells were involved in the anti-tumor immune responses. Vaccination was associated with induction of a sustained complete response in one patient and correlated with the generation of a potent anti-tumor T cell response. The remaining 10 patients progressed after a median duration of 8 months. We conclude that liposomal delivery of lymphoma membrane proteins is safe, induces tumor-specific CD4+ and CD8+ T cell responses, and may serve as a model for vaccine development against other human cancers. The induction of clinical response warrants further testing of this novel formulation in the setting of MRD where the immunosuppressive effects of the tumor are likely to be least.

Cross-reactive recognition of human and primate cytomegalovirus sequences by human CD4 cytotoxic T lymphocytes specific for glycoprotein B and H.

Although the importance of CD4+ T cell responses to human cytomegalovirus (HCMV) has recently been recognized in transplant and immunosuppressed patients, the precise specificity and nature of this response has remained largely unresolved. In the present study we have isolated CD4+ CTL which recognize epitopes from HCMV glycoproteins gB and gH in association with two different HLA-DR antigens, DRA1*0101/DRB1*0701 (DR7) and DRA1*0101/DRB1*1101 (DR11). Comparison of amino acid sequences of HCMV isolates revealed that the gB and gH epitope sequences recognized by human CD4+ T cells were not only conserved in clinical isolates from HCMV but also in CMV isolates from higher primates (chimpanzee, rhesus and baboon). Interestingly, these epitope sequences from chimpanzee, rhesus and baboon CMV are efficiently recognized by human CD4+ CTL. More importantly, we show that gB-specific T cells from humans can also efficiently lyse peptide-sensitized Patr-DR7+ cells from chimpanzees. These findings suggest that conserved gB and gH epitopes should be considered while designing a prophylactic vaccine against HCMV. In addition, they also provide a functional basis for the conservation of MHC class II lineages between humans and Old World primates and open the possibility for the use of such primate models in vaccine development against HCMV.

The use of protein structure/activity relationships in the rational design of stable particulate delivery systems.

The recombinant heat shock protein (18 kDa-hsp) from Mycobacterium leprae was studied as a T-epitope model for vaccine development. We present a structural analysis of the stability of recombinant 18 kDa-hsp during different processing steps. Circular dichroism and ELISA were used to monitor protein structure after thermal stress, lyophilization and chemical modification. We observed that the 18 kDa-hsp is extremely resistant to a wide range of temperatures (60% of activity is retained at 80 degrees C for 20 min). N-Acylation increased its ordered structure by 4% and decreased its beta-T1 structure by 2%. ELISA demonstrated that the native conformation of the 18 kDa-hsp was preserved after hydrophobic modification by acylation. The recombinant 18 kDa-hsp resists to a wide range of temperatures and chemical modifications without loss of its main characteristic, which is to be a source of T epitopes. This resistance is probably directly related to its lack of organization at the level of tertiary and secondary structures.