R. Nayak

Perpetuation of immunological memory: a relay hypothesis

A mechanism is proposed which explains the perpetuation of B‐cell immunological memory indefinitely without requiring the presence of long‐living memory cells or persisting antigen. The salient feature of this model is that immunological memory can be perpetuated indefinitely through the mutual interaction of idiotypic and anti‐idiotypic B cells. These cells mutually stimulate and clonally expand with either specific or bystander T‐cell help. Because B cells can present antigen, they present ‘apparently foreign’ idiopeptides to T cells. The idiopeptides of de novo synthesized antibody is presented to CD8+ T cells that recognize the idiopeptide‐presenting cell as targets and regulate their population. The recycling of immunoglobulins from surface to endosomal compartment of B cells leads to the presentation of idiopeptides by major histocompatibility complex (MHC) class II to CD4+ T cells. Even if the majority of the clonally expanded cells die because of lack of stimulation, cytotoxic T lymphocyte (CTL) lysis or for other reasons, the surviving cells will be able to carry forward the memory. This mechanism also provides a means for affinity maturation through idiotypic selection of somatically mutated high affinity cells or those from the naïve pool. We have termed these two types of complementary B cells as Burnet B cells: those which recognize the antigen or antigen mimic, and Jerne B cells, which can recognize the idiotypes of antibody and carry antigen mimics. The proposed hypothesis can explain differential duration of memory for different antigens, the shelf space paradox, affinity maturation, repertoire shift, etc.

Characterization of T-cell immunogenicity of two PE/PPE proteins of Mycobacterium tuberculosis

The PE and PPE proteins of Mycobacterium tuberculosis form a source of antigenic variation among different strains of this bacterium. Two of the PE_PGRS protein-encoding genes, rv3812 and rv3018c, are expressed in pathogenic mycobacteria and are implicated, respectively, in the persistence of the organism in macrophages and in virulence. Peptides derived from these proteins have been predicted to bind major histocompatibility complex (MHC) class I with high affinity on the basis of immunoinformatics analysis, suggesting a possible role for these proteins in antimycobacterial immunity. In the present work, using DNA constructs containing the rv3812 and rv3018c genes of M. tuberculosis, the immunogenicity of these proteins was demonstrated in BALB/c mice. Immunization with either DNA construct induced a significant number of CD8+-type T cells and a strong Th1-type response, with high gamma interferon (IFN-gamma) and low interleukin-4 responses. Three nonameric peptides of Rv3812 and two of Rv3018c elicited a strong T-cell response in an MHC-restricted manner. An epitope-specific response was demonstrated by the lysis of peptide-pulsed antigen-presenting cells, release of perforin and IFN-gamma production. Experimentally, these peptides bound with high affinity to MHC H-2Kd and showed low dissociation rates of peptide-MHC complexes. This study suggests that the identified T-cell epitopes may contribute to immunity against tuberculosis if included in a vaccine.

Detection of interferon gamma-secreting CD8+ T lymphocytes in humans specific for three PE/PPE proteins of Mycobacterium tuberculosis

The PE and PPE family of proteins of Mycobacterium tuberculosis have been hypothesized to play important roles in the biology of the organism and some proteins have been shown to be involved in eliciting T-cell responses. Earlier, we had identified putative HLA class I binding epitopes of the PE and PPE proteins of Mycobacterium tuberculosis employing computational and molecular modeling approaches. In the present work, three of the PE/PPE family proteins, coded by Rv1818c, Rv3812 and Rv3018c genes, were selected based on the computational analysis for testing human immune responses. PBMCs from patients with active tuberculosis and healthy, BCG vaccinated, PPD-positive individuals were tested for in vitro proliferative response and gamma-interferon production using synthetic peptides derived from the chosen proteins. Significant differences were seen in the responsiveness between healthy controls and patients. Antigen-specific T-cell lines were established from the PBMCs of healthy controls and their responses to peptide-specific CD8+ T-cell effectors were shown to be present at high frequency in the PBMCs of PPD+ controls. The T-cell lines also showed cytotoxic activity against the peptide pulsed monocytes.

Recombinant hemagglutinin protein of rinderpest virus expressed in insect cells induces humoral and cell mediated immune responses in cattle

Rinderpest virus causes a highly contagious and often fatal disease in domestic and wild ruminants. The surface glycoproteins, hemagglutinin (H) and fusion (F) proteins of this enveloped virus are known to confer protective immunity in cattle. We have reported the generation of a recombinant baculovirus expressing H protein and studied its protective properties in cattle. In this report, we demonstrate that the recombinant baculovirus encoded H protein expressed in insect cells gets incorporated into extracellular baculovirus. Single administration of low doses of purified recombinant extracellular virus with or without adjuvant induces virus neutralizing antibody responses and bovine leukocyte antigen (BoLA) class II restricted helper T cell responses in cattle.

Maintenance of antigen-specific immunological memory through variable regions of heavy and light chains of anti-idiotypic antibody

Immunological memory is characterized by a quick and enhanced immune response after re‐exposure to the same antigen. To explain the mechanism involved in generation and maintenance of immunological memory, we had earlier proposed a hypothesis involving the relay of memory by idiotypic and anti‐idiotypic B cells. The peptidomimic present in the hypervariable region of anti‐idiotypic antibody was hypothesized to carry forward immunological memory. In the present work, we provide evidence supporting a role for the anti‐idiotypic antibody in eliciting antigen‐specific B‐cell and T‐cell responses. Employing the idiotypic monoclonal antibody (Ab1) specific for haemagglutinin (H) protein of rinderpest virus, Ab2β was generated, which possesses an internal image of the H protein in the region between amino acids 527 and 556. We demonstrate that antigen‐specific memory is perpetuated by immunization with Ab2, as shown by maintenance of antigen‐specific T‐cell responses upon restimulation in vitro of Ab2 immune splenocytes by antigen‐presenting cells expressing H protein or pulsed with H‐protein‐derived peptides. We have also shown that boosting with antigen‐specific anti‐idiotypic B cells generates a memory response in antigen‐primed mice. Evidence has been provided for the existence of an antigen‐specific B‐cell idiotypic network in the body that supports the perpetuation of immunological memory as proposed in the relay hypothesis.

Activated mouse T cells downregulate, process and present their surface TCR to cognate anti-idiotypic CD4 + T cells

The ability of activated T cells to present foreign antigens through the MHC class II pathway has been shown in the case of human, rat and mouse T cells. In the present study, the ability of activated T cells to present their endogenous TCR in association with MHC class II molecules to CD4+ T cells was shown. Upon activation mouse T cells downregulate their surface TCR, which are degraded into peptides in endosomal/lysosomal compartments. The idiopeptides (peptides derived from the variable region of the TCR) are presented to cognate anti‐idiotypic CD4+ T cells, resulting in activation and proliferation of these cells. Interaction of idiotypic and anti‐idiotypic T cells brought about by presentation of TCR idiopeptide may have important implications for T‐cell vaccination and perpetuation of T‐cell memory not requiring persisting antigen or long‐lived memory cells.

Inhibition of replication of rinderpest virus by 5-fluorouracil

5-Fluorouracil (5FU), an analogue of uracil, was found to inhibit the production of infectious particles of rinderpest virus (RPV) in Vero cells (African green monkey kidney cells) by 99%, at a concentration of 1 microgram/ml. The levels of individual mRNA specific for five of the virus genes were also reduced drastically, while the level of mRNA for a cellular housekeeping gene-glyceraldehyde-3-phosphate dehydrogenase (GAPDH)-was unaltered by fluorouracil treatment of infected cells. Both virus RNA and protein synthesis showed inhibition in a dose-dependent manner. The virions which budded out of 5-fluorouracil-treated cells also contained reduced amounts of virus proteins compared with virus particles from untreated cells.

Idiotype and anti-idiotype specific T cell responses on transplantation with hybridomas reactive to viral hemagglutinin and human tumor antigen

B cell hybridomas expressing class I and II MHC molecules and producing antibodies directed against hemagglutinin protein of Rinderpest virus and human Mucin-1 have been used as surrogate B cells to study T cell responses against the antigens. The observed CTL and lymphoproliferative response indicates that anti-idiotypic B cells termed Jerne cells stimulate both T helper and T cytotoxic cells by virtue of their ability to present recycled or regurgitated peptido-mimics of antigen to T helper cells through class II MHC and de novo synthesized peptido-mimics of antigens to CTLs. Thus, T cell memory response can be perpetuated by anti-idiotypic Jerne B cells and these findings lend support to the earlier proposed relay hypothesis for perpetuation of immunological memory (IM).

Recombinant Hemagglutinin Protein of Rinderpest Virus Expressed in Insect Cells Induces Cytotoxic T-Cell Responses in Cattle

Rinderpest virus (RPV), a member of the genus Morbillivirus within the Paramyxoviridae family, causes a highly contagious and often fatal disease known as rinderpest in wild and domestic ruminants. The envelope of the virus contains two surface glycoproteins, namely the hemagglutinin (H) and the fusion (F) proteins, both of which have been shown to confer protective immunity in animals. In this paper, we demonstrate that single administration of low doses of recombinant H protein of RPV expressed in insect cells in the form of extracellular virus induces long lasting bovine leukocyte antigen class I restricted cytotoxic T-cell (CTL) responses in cattle in the absence of adjuvant. This is the first report of CTL responses in cattle against one of the protective antigens of RPV.

Mapping of T-helper epitopes of Rinderpest virus hemagglutinin protein.

Rinderpest virus (RPV) is a highly contagious and often fatal disease of domestic and wild ruminants, caused by rinderpest virus of the genus Morbillivirus under the family Paramyxoviridae. Hemagglutinin (H) and fusion (F) proteins of this enveloped virus confer protective immunity against experimental challenge with virulent rinderpest virus. We have earlier demonstrated that immunization with a single dose of recombinant extracellular baculovirus expressing H protein elicits H-specific humoral and lymphoproliferative responses in cattle. The lymphoproliferative responses are predominantly BoLA class II restricted. In this work, we have analyzed lymphoproliferative responses of peripheral lymphocytes from immunized cattle to truncated H protein fragments expressed in E. coli for locating domains harboring Th epitopes. One region (aa 113-182) recognized by immune T cells is conserved in the H protein of measles virus, which was earlier shown to contain a dominant Th epitope in mouse. Synthetic peptides within this region of measles virus H protein were used to identify a Th epitope conserved in the H protein of RPV virus (aa 123-137) in cattle. A second Th epitope located at the C-terminus of RPV-H was mapped to the region corresponding to aa 512-609 using truncated protein fragments expressed in E. coli. The C-terminal epitope (aa 575-583) was mapped using synthetic peptides corresponding to measles virus H as well as RPV-H protein.