Thesis (Ph.D. in Immunology) -- University of Colorado, 2006. / Typescript. Includes bibliographical references (leaves 277-285). Free to UCDHSC affiliates. Online version available via ProQuest Digital Dissertations;

Contraception through a vaccine has been a very attractive proposition and several attempts were made in the past. To achieve contraception through immunological means, several points need to be considered. First, the targeted antigen should be an important component of reproduction and interference in its actions should lead to infertility. Second, the antigen must be highly immunogenic and the antibodies elicited should be able to block the functions of the antigen. Third, the antibody titres should be effective and must sustain for longer periods. Gonadotropins fulfill all the above criteria and therefore, have been attractive targets for developing human contraceptive vaccines. The pituitary gonadotropins- Luteinizing hormone (LH) and the Follicle stimulating hormone (FSH) are the principal regulators of the reproduction process in all the mammalian species (McLachlan et al., 1995c; Moudgal et al., 1992b; Murty et al., 1979a; Selvaraj and Moudgal, 1994a; Weinbauer et al., 1991). In males, LH binds to its specific receptor-LHR, expressed on the Leydig cells and regulates the production of testosterone. This testosterone binds to the androgen receptors expressed in the Sertoli cells and along with FSH, which binds to the specific receptors present on the Sertoli cell membranes, regulate the testicular functions and the spermatogenesis (Simoni et al., 1997; Themmen and Huhtaniemi, 2000; Ulloa-Aguirre and Timossi, 1998). The well documented studies have unequivocally established that the specific immunoneutralization of either hormone by active or passive immunization, leads to disruption of the gonadal functions (Fraser et al., 1986a; Marathe et al., 1995; Moudgal et al., 1992b; Murty et al., 1979b; Shetty et al., 1996; Srinath et al., 1983b) and consequent infertility and this observation formed the basis of the human contraceptive vaccines (Moudgal et al., 1997b; Talwar et al., 2011a; Talwar et al., 2009a). Several studies using testosterone as the main male hormonal contraception method (Matsumoto et al., 1986; Matsumoto et al., 1983a) and anti-hCG vaccine as the female hormonal contraceptive vaccine reached Phase I and II clinical trials (Talwar, 1997; Talwar et al., 1994; Talwar et al., 1997) . However, these human contraceptive vaccines faced several limitations. There was a need to inhibit only particular segments of the entire reproduction process whereas others needed to remain completely unaffected. For example, in males, the FSH regulated functions, the sperm production and spermatogenesis needed to be inhibited whereas the LH/testosterone associated functions should be unaffected. Similarly in females, the functions of hCG alone, elaborated by the conceptus should be blocked without affecting either LH or FSH regulated functions, thus, maintaining the normal reproductive cycle. This however is a difficult task especially when the antigens share a large degree of homology and common subunits (Pierce and Parsons, 1981). Moreover, the issues relating to the development and sustenance of high titres of the bioneutralizing antibodies were major limitations of these human contraceptive vaccines. Therefore, despite reaching Phase I and II clinical trials, these studies did not progress further. However, the same concept of an immunocontraceptive vaccine involving the neutralization of the functions of the gonadotropins is an extremely attractive strategy for controlling the animal populations where the reproduction process could be inhibited in its entirety. The overgrowing populations of the stray animals such as dogs and cats pose problems unlike those experienced with the human overpopulation. Thus, there is an immediate need to develop the methods of controlling the populations of these animals both in the developed and the developing countries. Whereas, in countries like the US, the major emphasis is on the domestic animals, in countries like India, the populations of the stray animals need to be controlled. The current methods employed for reducing the numbers of these animals include either castration or culling of the animals. These methods are however, traumatic, unsafe and not widely accepted by the society. The animal contraceptive vaccines currently available are mostly GnRH vaccines which have high cost of production, are not safe for animal use and elicit unwanted side effects. Apart from these, the animals need multiple administrations of these vaccines to elicit high and effective antibody titres, mostly with the use of conventional but non-approved adjuvants (Boedeker et al., 2009; McCoy, 1994). As mentioned above, the gonadotropins, by virtue of their ability to control the mammalian reproduction process, are attractive targets for achieving contraception. Moreover, the ease of administration of this vaccine to neutralize the functions of the endogenous circulating hormones makes them ideal targets for developing animal immunocontraceptive vaccines. This method of neutralizing the functions of the gonadotropins is also humane and safe for the animals as opposed to the current methods which are employed to reduce their numbers. However, in case of animal contraception, particularly for strays such as dogs, where large numbers of animals need to be treated, the challenge is to develop a method to sustain the high levels of the bioneutralizing antibodies for prolonged periods preferably with a single administration of the immunogen and without the use of conventional adjuvants such as the Freund’s adjuvant. In the present study, an attempt has been made develop a strategy to achieve a sustained immune response to small quantities of the hormonal antigens, preferably with a single administration of the immunogen resulting in complete disruption of the gonadal function for prolonged periods. To achieve this goal, recent developments in the field of immunology and vaccinology have been employed. This involves targeting of the hormonal antigens to the dendritic cells. Targeting the antigens to the dendritic cells for vaccination is becoming an extremely fascinating strategy and is being used extensively to target the antigens involved in several diseases (Escudier et al., 2005; Frankel et al., 1998; Garcia et al., 2005; Nouri-Shirazi et al., 2000a; Nouri-Shirazi et al., 2000b; Steinman and Germain, 1998). Most antigens are targeted to the dendritic cells by coupling them to the antibodies specific for the receptors expressed on the dendritic cell surface. One such receptor is the DEC205, which is expressed on most of the dendritic cells (Jiang et al., 1995) and is being widely used to develop vaccines and vaccination strategies. Targeting the antigens to the dendritic cells provides advantages such as ability to induce hundred fold higher immune response to very low doses of antigen without the use of any conventional adjuvant (Bonifaz et al., 2004a). Therefore, in the present study, these features of the dendritic cells have been harnessed to target the hormonal antigens (hCG and hFSH) to the canine DEC205 receptor to induce a long-term immune response capable of disrupting the gonadal functions. Towards this goal of delivering hormonal antigens to the dendritic cells, a fragment of the canine DEC205 corresponding to the Cysteine Rich Fibronectin II domain (CR/FNII) was expressed and used to isolate several canine DEC205 specific recombinant antibodies in the form of single chain fragment variable (ScFvs) from the Tomlinson’s and the yeast human ScFv display libraries. From a pool of eight unique ScFvs screened from the Tomlinson’s libraries, three ScFvs namely B3, G10 and H4 were characterized. All these ScFvs could bind to the human DEC205 receptor but not to the mouse DEC205. Their inability to recognise the mouse DEC205 suggested that mouse could not be used as the model system for these studies and therefore, a surrogate model system was needed. As the canine CR/FNII shared a high degree of homology with the rabbit counterpart, adult rabbits have been used as the surrogate model for immunization studies after confirming the binding of the ScFvs to the rabbit dendritic cells. Since the goal of the study was to deliver the hormonal antigens to the dendritic cells, each ScFv was translationally fused to a core streptavidin fragment, thus creating bi-functional agents (ScFv-CS) capable of binding to the dendritic cells and also to any biotin-tagged antigen, thus delivering the antigen to the dendritic cells. Of the three ScFvs, the ScFv-CS-H4 which could bind to the canine CR/FNII with the KD of 25nM was used for demonstrating the ability of the ScFv-hormone complex to elicit the bioneutralizing antibody response. The ScFv-CS-H4-biotin-hCG or hFSH or both were administered to adult male rabbits along with poly IC: LC, a Toll-like receptor agonist and the antibody titres were monitored. It was possible to maintain high titres of the bioneutralizing antibodies for more than one year with a single administration of the immunogen. Testicular histology of the immunized animals showed extensive disruption of spermatogenesis with most of the germ cells being TUNEL positive undergoing apoptosis. There was complete absence of elongated spermatids and sperms in the testis indicating infertility caused by immunization with the gonadotropins. These data show that targeting the hormonal antigens to the dendritic cells leads to long-term infertility with minimal immunization. Although the ScFvs from the Tomlinson’s libraries were able to deliver the hormonal antigens to the dendritic cells and produce robust and sustained antibody response capable of disrupting the gonadal functions, the affinities of these ScFvs to DEC205 were moderate. It was felt that increasing the affinities of the ScFvs could enhance the effect with respect to the dose of the antigen that needs to be administered and the duration until which the high antibody titres could be maintained. Therefore, the yeast human ScFv display library offering higher diversity of the human ScFvs displayed, was screened for high affinity DEC205 specific binders. From a pool of several ScFvs, six unique ScFvs were characterized. The amino acid sequences of all ScFvs followed the Kabat's rules for identifying the complimentarity determining regions of the heavy and the light chains of the antibodies. All these ScFvs were unique in their amino acid sequences. The dissociation constants of all these antibodies for the canine CR/ FNII ranged from 10-9 to 10-11 M which was 20-300 fold higher than the ScFvs obtained from the Tomlinson’s libraries. The best ScFv obtained from this library was ScFv-92 with a KD value of 8 x10-11 M. All these ScFvs were able to deliver the payload antigen to both, the mouse DEC205 over-expressing cells and the bone marrow derived dendritic cells. Mice immunized with yeast display ScFvs also yielded antibody response to very small quantities of the immunogen with the highest antibody titres obtained with the ScFv-92. It was further demonstrated that all ScFvs also activated the cell-mediated immunity with significant increase in the antigen stimulated T cell proliferation. These ScFvs could also deliver the antigen to the human dendritic cells differentiated from the human monocytes in vitro, thus emphasising their utility in human vaccine development. An attempt was also made to develop nanoparticle (NP) based strategies of delivering the antigen to the dendritic cells. The PLGA-NPs, encapsulating hCG and coated with the DEC205 ScFv-92 was able to elicit high antibody response to very low doses of the antigen. This response could be sustained for 120 days and was higher than the response obtained with similar doses of hCG encapsulated NPs or hCG complexed to ScFv-92 alone. Targeting of the NPs also elicited antigen specific T cell response thus, potentiating their use in cell mediated immunity along with humoral immune responses. In conclusion, this approach of delivering the gonadotropins to the dendritic cells resulted in the production of bioneutralizing antibodies that could disrupt the gonadal functions for a prolonged period and can be effectively used in the fields for controlling the animal populations. This method fulfils all the criteria for any animal contraception. This strategy also elicits both T cell mediated and humoral immunity and can thus be used for producing vaccine against viral and parasitic infections. It can also be used for cancer immunotherapy. Another exciting feature of the strategy used in this study is the usage of ScFv-CS which allows the delivery of any biotin tagged antigen to the rodent and human dendritic cells. As discussed above, the methods for controlling the animal populations are expected to be effective, humane, safe, simple, non-surgical, single shot with long lasting effects, cheap, applicable in the fields and widely accepted by different societies. The methods presented in this study fulfill all these criteria and should be effective in controlling populations of different animal species.

The immune response to an infection is determined by a number of factors, which also affect the generation of memory T cells afterwards. The immune response can also affect the stability of the pre-existing memory populations. The memory developed after an infection can influence the response to subsequent infections with unrelated pathogens. This heterologous immunity may deviate the course of disease and alter the disease outcome. The generation and stability of memory CD8 T cells and the influence of the history of infections on subsequent heterologous infections are studied in this thesis using different viral infection sequences. Previous studies using mice lacking individual immunoproteasome catalytic subunits showed only modest alterations in the CD8 T cell response to lymphocytic choriomeningitis virus (LCMV). In this study, I found that the CD8 T cell response to LCMV was severely impaired in mice lacking all three catalytic subunits of the immunoproteasome, altering the immunodominance hierarchy of the CD8 T cell response and CD8 T cell memory. Adoptive transfer experiments suggested that both inefficient antigen presentation and altered T cell repertoire contribute to the reduction of the CD8 T cell response in the immunoproteasome knockout mice. Immune responses generated during infections can reduce pre-existing memory T cell populations. Memory CD8 T cells have been shown to be reduced by subsequent heterologous infections. In this study, I re-examined the phenomenon using immune mice infected with LCMV, murine cytomegalovirus (MCMV) and vaccinia virus (VACV) in different infection sequences. I confirmed that memory CD8 T cells were reduced by heterologous infections, and showed that LCMV-specific memory CD4 T cells were also reduced by heterologous infections. Reduction of the memory CD8 T cells is thought to be the result of apoptosis of memory CD8 T cells associated with the peak of type I interferon early during infection. I showed that memory CD4 T cells were similarly driven to apoptosis early during infection; however, Foxp3+ CD4+ regulatory T cells were relatively resistant to virus infection-induced apoptosis, and were stably maintained during LCMV infection. The stability of Treg cells during viral infections may explain the relatively low incidence of autoimmunity associated with infections. The history of infections can deviate the course of disease and affect the disease outcome, but this heterologous immunity is not necessarily reciprocal. Previous studies have shown the effects of heterologous immunity during acute infections. In this thesis, I showed that the history of LCMV infection led to higher viral titers during persistent MCMV infection, caused more severe immunopathology at the beginning of infection, and reduced the number of MCMV-specific inflationary memory CD8 T cells after the period of memory inflation. In a different context of infection, the history of LCMV infection can be beneficial. LCMV-immune mice have been shown to have lower viral titers after VACV infection, but VACV-immune mice are not protected during LCMV infection. I found that memory CD8 T cells generated from LCMV and VACV infections were phenotypically different, but the differences could not explain the nonreciprocity of heterologous immunoprotection. By increasing the number of crossreactive VACV A11R198-205-specific memory CD8 T cells, however, I showed that some VACV-immune mice displayed reduced viral titers upon LCMV challenge, suggesting that the low number of potentially cross-reactive CD8 T cells in VACV-immune mice may be part of the reasons for the non-reciprocity of immunoprotection between LCMV and VACV. Further analysis deduced that both number of potentially cross-reactive memory CD8 T cells and the private specificity of memory CD8 T cell repertoire played a part in determining the outcome of heterologous infections.

Thesis (MScMed (Biomedical Sciences. Molecular Biology and Human Genetics))--University of Stellenbosch, 2006. / The ESAT-6 gene cluster regions are duplicated 5 times in the genome of Mycobacterium tuberculosis. ESAT-6 gene cluster region 1 is the most frequently studied region as it contains RD1 (region of difference 1). RD1 is a 9.5 Kb deletion region confirmed to be involved in mycobacterial virulence and pathogenesis, and is present in virulent M. bovis strains, yet absent in all attenuated M. bovis BCG vaccine strains. The antigens CFP-10 and ESAT-6, which both evoke strong T-cell responses in experimental animals and humans, are situated in the RD1 region, and are thought to be key antigens in mycobacterial virulence. The absence of this region from the genomes of all BCG vaccine strains, led to the conclusion that the mechanism of attenuation of M. bovis BCG was due to the loss of RD1. Studies have shown that this attenuation is attributed to the loss of cytolytic activity mediated by secreted ESAT-6 (and some of the genes responsible for its secretion), which in turn results in reduced tissue invasiveness. The potent T-cell antigens ESAT-6 and CFP-10 are secreted without ordinary sec-dependent secretion signals. A study of the potential functions of the proteins encoded by the ESAT-6 gene clusters shows that most of these proteins have a potential to function in a protein-dependent ATP-binding cassette active transport system. It has been shown that ESAT-6 gene cluster region 1 is responsible for the secretion of the ESAT-6 and CFP-10 genes contained in this region, explaining the absence of any ordinary sec-dependent secretion signals in the amino acid sequences of members of this family. In order to elucidate the regulation of expression of the ESAT-6 gene cluster region 1, shown to encode for a secretion system for ESAT-6 and CFP-10 and to be involved in virulence, an operon analysis and promoter identification experiments were carried out in this study. The analysis of the ESAT-6 gene cluster region 1 showed the existence of more than one operon in this region and three constitutively-expressed promoters driving the expression of the genes in the operons. These results provide insight into the functional relationship (regulatory and secretory mechanisms) between the genes contained within ESAT-6 gene cluster region 1.None of the other four ESAT-6 gene cluster regions have been proven to also encode secretion systems. Preliminary studies indicated that the ESAT-6 gene cluster region 3 is expressed in its entirety as one single operon and a strong promoter involved in the expression of this region was identified. Mtb9.9A (the ESAT-6 antigen of the ESAT-6 gene cluster region 5) have also been shown to evoke strong T cell responses and to be secreted without any ordinary secretion signal. During the present study, we thus aimed to investigate the secretion of Mtb9.9A in order to determine whether it is also secreted by a dedicated secretion system encoded by ESAT-6 gene cluster region 5. The fact that region 5 was shown to be the last of the four duplications is important, as a positive result with this region would indicate whether the other four gene clusters share a similar secretion function. ESAT-6 gene cluster regions 2, 4 and 5 were isolated in the present study to form part of subsequent ESAT-6 gene cluster region secretion studies. Mtb9.9A was cloned, expressed and purified for antibody-generation, Resulting antibodies were used in an antigen secretion analysis. The secretion analysis entailed the integration of the isolated ESAT-6 gene cluster region 5 into the genome of M. smegmatis and investigation of the influence of the genes (contained in region 5) on the secretion of a heterologously expressed Mtb9.9A-HA-tagged fusion protein. We therefore attempted to show whether the proteins encoded by the ESAT-6 gene cluster region 5 also function together as a mycobacterial membrane-bound complex involved in protein-dependent transport and if so, whether this transport system is responsible for the active secretion of the native ESAT-6 antigen (designated Mtb9.9A) of region 5. This study opens the way for the understanding of the regulation, transport- and secretion mechanisms of important T-cell antigens of the mycobacteria, thereby giving insight into and building onto our understanding of the pathogenicity of Mycobacterium tuberculosis. A better understanding of these mechanisms could lead to the development of efficient strategies to either terminate or enhance secretion of antigens, which in turn will have an impact on drug and vaccine design and development.

Thesis (PhD)--Stellenbosch University, 2002. / ENGLISH ABSTRACT: Pathogenic organisms frequently utilize proteases to perform specific functions related to virulence. There is little information regarding the role of proteolysis in Mycobacterium tuberculosis and no studies on the potential involvement of these enzymes in the pathogenesis of tuberculosis. The present study initially focused on the characterization of a family of membrane anchored, cell wall associated, subtilisin-like serine proteases (mycosins-1 to 5) of Mycobacterium tuberculosis. These proteases were shown to be constitutively expressed in M. tuberculosis, to be located in the cell wall of the organism and to be potentially shed (either actively or passively) from the wall. Relatively high levels of gamma interferon secretion by T-cells in response to these proteases were observed in Mantoux positive individuals. The absence of any detectable protease activity lead to a protein sequence analysis which indicated that the mycosins are probable mycobacterial-specific proprotein processing proteases. To identify possible substrates for these proteases, the genome sequence regions surrounding the mycosin genes were analyzed. This revealed that the mycosin genes are in fact part of a cluster of 6 to 12 genes which have been duplicated multiple times in the genome of M. tuberculosis. Due to the presence of members of the previously described ESAT-6 T-cell antigen family within this duplicated region, the five gene cluster regions were named the ESAT-6 loci. In silico analysis of finished and unfinished genome sequencing data revealed the presence of orthologues of the Mycobacterium tuberculosis H37Rv ESAT-6 loci in the genomes of other mycobacteria, e.g. M. tuberculosis CDC1551, M. tuberculosis 210, M. bovis, M. leprae, M. avium, and the avirulent strain M. smegmatis. Phylogenetic analyses done on the resulting sequences have established the duplication order of the gene clusters and demonstrated that gene cluster region 4 (Rv3444c-3450c) is ancestral. Region 4 is also the only region for which an orthologue could be found in the genomes of Corynebacterium diptheriae and Streptomyces coelicoior. Thus, the comparative genomic analyses revealed that the presence of the ESAT-6 gene cluster seems to be a unique characteristic shared by members of the high G+C gram-positive bacteria and that multiple duplications of this cluster have occurred and have been maintained only within the genomes of members of the genus Mycobacterium. The ESAT-6 gene cluster regions were shown to consist of the members of the ESAT-6 gene family (encoding secreted T-cell antigens that lack detectable secretion signals), the mycosins (secreted, cell wall-associated subtilisin-like serine proteases) as well as genes encoding putative ABC transporters, ATP-binding proteins, and other membrane-associated proteins. Thus, from the observation that members of the ESAT-6 family are secreted without the normal sec-dependent secretion signals, it was hypothesized that the membrane-associated and energy-providing proteins function together to form a transport system for the secretion of the members of the ESAT-6 protein family. Supporting this hypothesis, one of the ESAT-6 gene clusters was shown to be expressed as a single polycistronic RNA, forming an operon structure. The promoter for this operon, P e s r e g 3. was also identified and its activity characterized. Subsequent secretion analyses results have shown that secretion of members of the ESAT-6 protein family is dependent on the presence of the proteins encoded by the ESAT-6 gene cluster regions, confirming the putative transport-associated functions of the ESAT-6 gene cluster-encoded proteins. The mycobacterial ESAT-6 gene clusters contain a number of features of quorum sensing and lantibiotic operons, and an extensive review of the literature have led to the hypothesis that the members of the ESAT-6 family may be secreted as signaling molecules and may be involved in the regulation of expression of genes during intracellular residence of the bacterium. In the final part of this study, the evolutionary history of the PE and PPE gene families (members of which is found situated in the ESAT-6 gene clusters) were investigated. This investigation revealed that the expansion of these families are linked to the duplications of the ESAT-6 gene clusters, which is supported by the absence of the multiple copies of the PE and PPE families in the genome of the fast-growing mycobacterium M. smegmatis. Furthermore, dot blot analyses showed that the PPE gene present in ESAT-6 gene cluster region 5 is able to distinguish between mycobacteria belonging to the slow-growing or fast-growing species, indicating a function for the genes of these two families and/or the ESAT-6 gene clusters in the phenotypical differences distinguishing these two groups of mycobacteria. In conclusion, this study has highlighted numerous important aspects of mycobacterial genomics and has greatly contributed to the current body of knowledge concerning the role of proteases, gene duplication and mechanisms of antigen expression and secretion in M. tuberculosis. / AFRIKAANSE OPSOMMING: Sien asb volteks vir opsomming

published_or_final_version / Pathology / Master / Master of Philosophy

Existe un grupo de enteropatía conocidas como AVSN que pueden simular enfermedad celíaca. Objetivo: El objetivo de este estudio es describir los hallazgos histológicos y de inmunohistoquímica en pacientes con enfermedad celíaca y AVSN. Material y métodos: 15 biopsias de pacientes con enfermedad celíaca y 19 biopsias con AVSN fueron reexaminados. Se estudió características histológicas tales como atrofia severa, hiperplasia de criptas, número de células plasmáticas, número de eosinófilos y presencia de neutrófilos. Asimismo, a través de inmunohistoquímica se estudió la presencia de linfocitos CD4, CD8, CD3, CD56. Resultados: Se encontró diferencia significativa en la mayor presencia de hiperplasia de criptas (p=0,0348) y mayor número de células plasmáticas (p=0,0348) en las biopsias de enfermedad celíaca que en las catalogadas como AVSN. El número de linfocitos CD8, CD4, CD56 y su distribución fue similar en ambos grupos. El porcentaje de linfocitos intraepiteliales CD3 positivos (p=0,0144) fue mayor en pacientes con AVSN. Conclusión: Los hallazgos histológicos e inmunohistoquímicos muestran más similitudes que diferencias. La diferencia hallada en nuestro estudio sugiere mayor respuesta inmune humoral en pacientes con enfermedad celiaca que en AVSN. / There is a group of enteropathies recently known as seronegative villous atrophy (SNVA), which can simulate celiac disease. Objective: The aim of this study was to describe histological and immunohistochemical differences between a group of Celiac disease and SNVA patients. Material and methods: Microscopy reexamination and Immunohistochemistry study were performed for a group of 15 celiac patients and 19 SNVA patients. Histological features as severe atrophy, crypt hyperplasia, plasma cells number, eosinophils number, neutrophils presence were studied; CD4, CD8, CD3, and CD56 markers were studied through immunohistochemistry. Results: There was a significant difference between the frequency of observation of crypt hyperplasia (p=0.0348) and plasma cells (p=0.0348) in celiac disease patients than SNVA patients. In celiac disease was bigger. The number and distribution of CD 8, CD4 and CD56 lymphocytes was similar in both groups. The percentage of CD3 positive intraepithelial lymphocytes (p=0.0144) was higher in SNVA. Conclusion: Histological and immunohistochemical evaluation shows more similarities than differences. The differences found in this study suggest more humoral immune response in celiac disease than in SNVA.

The aim of this study was to design a resolution typing system for the HLA-B gene. This technique involves a one-step PCR reaction utilizing genomic DNA and sequence-specific primers to determine the specificity of each allele and to produce a larger primer data base ideal for serological analysis. The application of this technique to serological analysis can improve serology detection which is currently hindered by antibody cross-reactivity and the unavailability of useful typing reagents.

This study describes the development and validation of mutagenized cloned DNA constructs, which correspond to the polymorphic regions of the class II region of the HLA complex. The constructs were used to verify the allelic specificity of primers and probes in polymerase chain reaction (PCR)-based HLA typing assays such as Sequence Specific Primers (SSP) and Sequence Specific Oligonucleotide Probes (SSOP). The constructs consisted of the entire polymorphic region of exon 2 of class II HLA allele sequences that included primer annealing sites or probe hybridization sites. An HLA allele sequence was inserted into a plasmid, cloned, then mutagenized to match a specific HLA allele, and finally, the correct clone was verified by bidirectional sequencing of the insert. Thus, the construct created a cloned reference DNA sample for any specific allele, and can be used to validate the accuracy of various molecular methodologies.

The Hepatitis B core antigen (HBcAg) has been proposed to be an ideal candidate for use as an adjuvant due to its immunogenicity, and tolerance to manipulations such as insertions of epitopes or covalent attachment of ligands. HBcAg is a complex macromolecule containing protein and nucleic acid. We investigated the effect of the removal and reconstitution of nucleic acids upon its structure. It’s been shown that the RNA content of hepadnavirus core antigens can be reduced significantly, but not be completely removed. Following removal of some of the RNA, antigens retain the ability to bind added nucleic acids, in particular, "immune-enhancing" synthetic oligonucleotides without affecting the structure of antigen or disrupting its ability to spontaneously self-assemble into core particles. The removal and addition of nucleic acids was successfully applied to an altered woodchuck core antigen, with a nucleic acid-based malaria epitope addition, giving rise to a potential vaccine adjuvant platform for malaria.