Fetal immunization of baboons induces a fetal-specific antibody response.

Neonates face a high risk of infection because of the immaturity of their immune systems. Although the transplacental transfer of maternal antibodies to the fetus may convey improved postnatal immunity, this transfer occurs late in gestation and may fail to prevent in utero infection. Both fetal immunization and in utero exposure to antigen can result in a state of immunologic tolerance in the neonate. Tolerance induction of fetal and premature infant lymphocytes has become a paradigm for neonatal responsiveness. However, fetal IgM responses have been demonstrated to maternal immunization with tetanus toxoid and to congenital infections such as rubella, toxoplasma, cytomegalovirus and human immunodeficiency virus. Moreover, 1-week-old infants can respond to standard pediatric vaccination, and neonates immunized with polysaccharide antigens do not develop immunologic tolerance. Here, direct immunization of the baboon fetus with recombinant hepatitis B surface antigen produced a specific fetal IgG antibody response. No specific maternal antibody response was detected, eliminating the possibility of vertical antibody transmission to the fetus. Some infants also responded to later vaccinations with hepatitis B surface antigen, indicating that no immunological tolerance was induced by prior fetal immunization. These results characterize the ability of the fetal immune system to respond to in utero vaccination. We demonstrate that active fetal immunization can serve as a safe and efficient vaccination strategy for the fetus and neonate.

Enhancement of the immune response to hepatitis B surface antigen. In vivo administration of antiidiotype induces anti-HBs that expresses a similar idiotype.

BALB/c mice receiving antiidiotype antibodies before the injection of hepatitis B surface antigen (HBsAg) generated an enhanced anti-HBs response. Mice given antiidiotype antibodies in a soluble form induced predominantly IgM anti-HBs, whereas alum-precipitated antiidiotype produced primarily IgG anti-HBs. Injection of antiidiotype antibodies alone induced anti-HBs that inhibited a common interspecies anti-HBs idiotype-antiidiotype reaction and recognized the group-specific determinant of HBsAg. These data support the view that antiidiotype antibodies may modulate the immune response to an infectious viral agent.

Suppression of in vivo tumor formation induced by simian virus 40-transformed cells in mice receiving antiidiotypic antibodies.

This study characterizes four private idiotypes (Id) associated with monoclonal antibodies (mAb) to simian virus 40 (SV40) tumor antigen (T-Ag), and to a cellular protein, p53. Anti-Id recognized Id determinants associated with the antibody-combining site. BALB/c mice receiving a pool of anti-Id directed against mAb recognizing distinct amino and carboxyl terminal epitopes of T-Ag before receiving a tumorigenic dose of SV40-transformed cells showed suppression of tumor formation. Serum obtained from these mice before tumor challenge contained anti-anti-Id that failed to bind T-Ag. These data support the potential role of regulatory idiotopes in tumor immunity.

Characterization of PEDF: a multi-functional serpin family protein.

Pigment epithelium-derived factor (PEDF) is a 50 kDa secreted glycoprotein that belongs to the non-inhibitory serpin family group. PEDF has been described as a natural angiogenesis inhibitor with neurotrophic and immune-modulation properties; it balances angiogenesis in the eye and blocks tumor progression. The mechanisms underlying most of these events are not completely clear; however, it appears that PEDF acts via multiple high affinity ligands and cell receptors. In this review article, we will summarize the current knowledge on the biochemical properties of PEDF and its receptors, the multimodal activities of PEDF and finally address the therapeutic potential of PEDF in treating angiogenesis-, neurodegeneration- and inflammation-related diseases.

Antibody to hepatitis B virus induced by injecting antibodies to the idiotype.

Anti-idiotype reagents that recognize a common idiotype associated with antibody to hepatitis B surface antigen (anti-HBs) were used to induce anti-HBs in mice. The anti-idiotype-induced anti-HBs was found to recognize the group-specific a determinant of hepatitis B surface antigen and to express an interspecies idiotype. These findings suggest that anti-idiotypes may be useful as vaccines or vaccine primers.

Anti-idiotypic antibody vaccine for type B viral hepatitis in chimpanzees.

Anti-idiotypic antibodies (anti-Id) that contain an internal image component that mimics the surface antigen of hepatitis B virus (HBsAg) were used to immunize chimpanzees. Four injections of the rabbit anti-Id preparation elicited an antibody response to HBsAg (anti-HBs). The antibody specificity appeared to be against the anti-Id, since the anti-Id immunogen was shown to bind the chimpanzee anti-HBs. Two chimpanzees immunized with the anti-Id, along with two control animals that were either untreated or received a nonimmune rabbit immunoglobulin G preparation, were challenged with infectious hepatitis B virus. Both control chimpanzees developed clinical and serological characteristics consistent with an active hepatitis B virus infection, whereas the two anti-Id treated chimpanzees were protected from infection. Since chimpanzees provide a relevant model of a human response to hepatitis B virus immunization and infection, these results indicate that anti-Id preparations such as that described here might be candidates for vaccines against human diseases.

Antiserum to a synthetic peptide recognizes the HTLV-III envelope glycoprotein.

In a study performed to determine which regions of the human T-cell lymphotrophic virus type III (HTLV-III) may represent vaccine candidates to prevent the acquired immune deficiency syndrome (AIDS), a synthetic peptide corresponding to amino acid sequence 735 to 752 of the precursor envelope glycoprotein of HTLV-III was used to immunize rabbits. The resulting rabbit antiserum to the synthetic peptide specifically recognized the precursor envelope glycoprotein (gp160) of HTLV-III. Human sera positive for antibody to HTLV-III reacted with this peptide. These findings indicate that synthetic peptides can be used to induce an immune response directed against a native envelope glycoprotein epitope of HTLV-III. The data are discussed in terms of using synthetic peptides to identify antigenic determinants involved in the induction of protective immunity and possibly as vaccine candidates against the etiologic agent of AIDS.

Immune response to hepatitis B surface antigen: enhancement by prior injection of antibodies to the idiotype.

Anti-idiotype reagents that recognize a common idiotype associated with the combining site of antibodies to hepatitis B surface antigen (anti-HBs) were used to manipulate the immune response to hepatitis B surface antigen in BALB/c mice. The injection of antibodies to the idiotype before antigenic stimulation resulted in an increase in the number of cells secreting immunoglobulin M antibodies to hepatitis B surface antigen. Anti-HBs-secreting cells were also induced by administration of antibodies to the idiotype without subsequent antigen exposure. These findings indicate that the immune response to hepatitis B surface antigen in mice is regulated through an idiotype-anti-idiotype network.

Obstetric fistula in rural Ethiopia.

OBJECTIVES: To determine the prevalence of obstetric fistula in rural Ethiopia and identify the circumstances and barriers to care that enhance development of obstetric fistula and its health and social consequences. DESIGN: A cross-sectional study. SETTING: The study was conducted in seven out of eleven administrative regions of Ethiopia by visiting randomly selected houses in rural areas and identifying women who have or had obstetric fistula and interviewing them. RESULTS: A total of 19,153 houses were visited. Untreated fistula prevalence was about 1.5 per 1000 amounting to approximately 26,819 women. Most of the patients were young women who delivered for the first time. Marriage took place early in life mostly through family arrangements or abduction. The median number of days in labour was three to eight. CONCLUSION: Promotive measures such as increasing age at marriage, and identification and treatment of patients should be intensified. There is a great need in improving accessibility and affordability of basic and emergency obstetric services for rural communities. Curving the situation in the long run requires dealing with the problem of poverty and improvement in the status of women.

Comparison of humoral immune responses and tumor immunity in mice immunized with recombinant SV40 large tumor antigen and a monoclonal anti-idiotype.

We compared the humoral immune responses induced in BALB/c mice by immunization with recombinant SV40 large tumor antigen (T-ag) with those induced by a monoclonal anti-idiotype (anti-Id), designated 58D, that is specific for SV40 T-ag-induced Id network components. We also challenged immunized mice with a lethal dose of SV40-transformed cells to assess in vivo tumor immunity. Two biweekly immunization with either SV40 T-ag or anti-Id 58D induced humoral responses that recognized both SV40 T-ag and anti-Id 58D. Four biweekly immunizations with SV40 T-ag increased the antigen-specific antibody titers and decreased the response to anti-Id 58D, while four biweekly immunizations of anti-Id 58D increased antibody titers to both itself and SV40 T-ag. Comparison of specific T-ag epitope and idiotope specificities indicated that SV40 T-ag and anti-Id 58D immunization generated responses that recognized a similar epitope on SV40 T-ag and expressed a shared idiotope recognized by anti-Id 58D. SV40 T-ag immunized mice challenged with a lethal dose of SV40-transformed cells were completely protected and no tumors were observed. This is despite the fact that little or no SV40 T-ag-specific cytotoxic T-lymphocyte activity was detectable. In contrast, only 3 of 10 mice immunized with anti-Id 58D were protected from a lethal challenge. These results indicate that, although monoclonal anti-Id immunization can induce responses that recognize similar SV40 T-ag epitopes and express shared idiotopes associated with antibodies to SV40 T-ag, the recombinant antigen itself induces superior in vivo tumor immunity.

Protection against a lethal challenge with SV40-transformed cells by the direct injection of DNA-encoding SV40 large tumor antigen.

Plasmid DNA encoding the large tumor antigen (T- ag) of SV40 was used to actively immunize mice to assess the induction of SV40 T-ag-specific immunity. Mice were injected with the naked DNA i.m., and immune responses were compared to those elicited in mice immunized with the recombinant SV40 T-ag protein. Compared to immunization with the recombinant protein, naked DNA induced weak antibody responses to SV40 T-ag. No increase in natural killer cell activity was observed following either recombinant protein or nucleic acid vaccination. However, the recombinant SV40 T-ag failed to induce SV40 T-ag-specific CTL responses, whereas the plasmid DNA encoding SV40 T-ag elicited CTL activity specific for SV40 T-ag. The SV40 T-ag-specific CTL lysed in vitro only syngeneic target cells (H-2(d)) expressing SV40 T-ag, indicating that the CTL are MHC restricted. Both the recombinant protein and naked DNA preparations induced immune responses that were protective against a lethal challenge with syngeneic SV40-transformed cells. A comparison of recombinant protein versus nucleic acid immunization indicates that both humoral and cell-mediated immune responses may play a role in SV40 T-ag immunity. These data indicate that active immunization with genes encoding tumor-specific antigens may be an efficacious strategy for the induction of tumor immunity.

Strategies for developing vaccines against acquired immunodeficiency syndrome.

There exists an abundance of literature on the prospects for developing vaccines against the etiologic viral agent of acquired immunodeficiency syndrome (AIDS). Excellent reviews from a variety of investigators are available on this subject. It is our intention to review the literature relative to potential strategies for developing a successful AIDS vaccine and to impart some of our concerns on the different types of vaccine that are being tested. We have also focused on some vaccine strategies that have not received much attention in previous review articles. Finally, the role of animal models in assessing vaccine safety and efficacy against AIDS has been briefly described.

T cells and HIV-induced T cell syncytia exhibit the same motility cycle.

Ameboid cells ranging in complexity from Dictyostelium amebas to human polymorphonuclear leukocytes (PMNs) translocate in a cyclical fashion. Using computer-assisted motion analysis, we have analyzed the motility of human lymphocytes of the immortal SupT1 cell line and of a peripheral blood mononuclear cell population highly enriched for CD4-positive cells (CD4-enriched PBMCs) on four substrates--plastic, dehydrated rat tail collagen, hydrated rat tail collagen, and bovine aortic endothelium. In addition, we have analyzed the motility on these substrates of syncytia induced by human immunodeficiency virus (HIV) in cultures of both cell types. It is demonstrated that both SupT1 cells and CD4-enriched PBMCs exhibit a motility cycle with a period of 1.6 min that is independent of substrate, independent of average cell velocity, and similar to the periods of translocating Dictyostelium amebas and PMNs. More surprisingly, it is demonstrated that HIV-induced SupT1 and PBMC syncytia with volumes 10 to 100 times those of single cells exhibit the same motility cycle as their single-cell progenitors. These observations support the generality of the motility cycle in animal cells and, for the first time, demonstrate that the cycle is independent of cell size.

Molecular characterization and structural modeling of immunoglobulin variable regions from murine monoclonal antibodies specific for hepatitis B virus surface antigen.

We have characterized structurally the V regions of a set of murine monoclonal antibodies designated A1.2, A3.1, and A2.1, which recognize a group-specific epitope associated with hepatitis B virus surface antigen (HBsAg). The selection of these antibodies for this characterization was based on data which indicated that A1.2 and A3.1 recognize an overlapping epitope, while A2.1 recognizes a different group-specific epitope, on the HBsAg molecule. In addition, a conformation-dependent cross reactive Id is expressed on both A1.2 and A3.1, but not on A2.1. We have determined the primary sequence structures of these three monoclonal antibodies to HBsAg (anti-HBs), and have aligned them to evaluate V region sequence homology and identify potential regions of structural homology which provide a basis for the HBsAg epitope recognition and the cross reactive Id. Both A1.2 and A3.1 express VH regions which are highly homologous to the VH NP gene family (V186-2), both use members of the DSP2 D region gene family and utilize the JH 2 and JH 1 J gene segments, respectively. Alternatively, A2.1 is related to the VH J558 gene family and expresses a fusion of the DFL16.1 and DQ52 D gene regions in conjunction with the MH 1 gene segment. Each of these three monoclonal anti-HBs utilize light chains from the V kappa 21 and the J kappa 4 gene families. Primary amino acid sequence data were employed to construct computer generated models of the A1.2, A3.1, and A2.1 V regions to determine potential antigen combining site structures and the basis for the expression of the cross reactive Id. These results are discussed in terms of potential interaction sites with HBsAg and V region sites involved in Id expression.

SV40 large tumor antigen associated synthetic peptides define native antigenic determinants and induce protective tumor immunity in mice.

Synthetic peptides were utilized to define antigenic determinants on simian virus 40 (SV40) large tumor antigen (T-ag). Six synthetic peptides representing predicted B-cell epitopes on SV40 T-ag were used to immunize mice to compare the humoral immune responses and ascertain the ability of the peptide preparations to induce protective tumor immunity in vivo. Anti-peptide antibodies from BALB/c and C57BL/6 mice were examined for reactivity with SV40 T-ag by various immunologic assays. Antibodies from both strains to four of the peptides recognized recombinant SV40 T-ag by ELISA. However, T-ag recognition by anti-peptide antibodies differed when assessed by Western blot. Antibodies induced by the same four peptides in BALB/c mice recognized T-ag, whereas only three of the sex peptides induced antibodies in C57BL/6 mice capable of recognizing SV40 T-ag by Western blot. Flow cytometric analysis revealed that antibodies to peptides corresponding to T-ag amino acid residues 632-652 and 690-708 from BALB/c mice were able to recognize the surface of SV40 transformed cells, whereas five of the six peptides induced surface reactive antibodies in C57BL/6 mice. More important, peptides 632-652 and 690-708 elicited a protective immune response in BALB/c mice subsequently challenged with a lethal dose of syngeneic SV40 transformed cells. However, this tumor immunity was incomplete as only 50% of the mice survived the tumor challenge. These data indicate that antibodies induced by synthetic peptides corresponding to predicted B-cell epitopes on SV40 T-ag are capable of recognizing native and denatured determinants on T-ag. Furthermore, immune responses elicited by selected peptides partially protected BALB/c mice from a lethal tumor challenge.

Structural characterization of viral neutralizing monoclonal antibodies to hepatitis B surface antigen.

In this study we describe the viral neutralizing activity of murine monoclonal antibodies (MAb) specific for hepatitis B surface antigen (HBsAg). This viral neutralizing activity was assessed in vitro by employing Hepatitis Delta Virus (HDV) and human hepatocytes as target cells. To further characterize these viral neutralizing antibodies we generated a panel of anti-idiotypic (anti-Id) reagents and serologically characterized these antibodies for epitope specificity, Id specificity, and Id heterogeneity. Direct binding and competitive inhibition solid phase enzyme immunoassay have demonstrated that two murine MAb specific for HBsAg (anti-HBs), designated A1.2 and A3.1, recognize similar or overlapping epitopes on HBsAg, while monoclonal anti-HBs, designated A2.1 recognizes a unique HBsAg epitope. Further, Id analysis using monoclonal and polyclonal anti-Id reagents have identified both a private and a cross-reactive Id, respectively, on the anti-HBs, A1.2 preparation. The source of the idiotypic cross-reactivity between A1.2 and A3.1 has been identified, using Western blot analysis, to conformational determinants expressed by the heavy (H) and light (L) chains of these monoclonal anti-HBs. Lastly, the intrastrain antibody repertoire induced following HBsAg immunization was found to be relatively restricted in heterogeneity by clonotype analysis using isoelectric focusing and affinity immunoblot analysis. Interspecies variability in the anti-HBs response was observed based on epitope recognition using purified anti-HBs from a variety of species.

Sequence comparisons of non-human primate HIV-1 coreceptor homologues.

Infection of non-human primate peripheral blood mononuclear cells (PBMCs) in vitro with primary human immunodeficiency virus type 1 (HIV-1) isolates is extremely inefficient and often unattainable. The mechanism of resistance to infection by primary HIV-1 isolates in chimpanzee and baboon PBMCs is unknown. In this study, two HIV-1 coreceptors, CCR5 and CXCR4, were sequenced from chimpanzee and baboon PBMCs to determine if any sequence variations or mutations in these genes could be responsible for resistance to HIV infection. Primers were designed from the human coreceptor sequences and were able to amplify the CCR5 and CXCR4 genes from these non-human primate cells. No 32 base pair deletion (delta32) mutations were found in any of the non-human primate samples tested. CXCR4 sequence analysis showed chimpanzee and baboon share 99.7 and 98% nucleotide sequence homology and 100 and 98.9% amino acid sequence homology, respectively, compared to the human sequence. CCR5 sequence analysis demonstrated that chimpanzee and baboon share 99.6 and 98% nucleotide homology and 100 and 98% amino acid homology, respectively, with the human sequence. These data indicate that no variations in these coreceptor gene sequences exist that can explain the lack of susceptibility to infection with primary HIV-1 isolates in non-human primate PBMCs.

Anti-peptide reagent identifies a primary-structure-dependent, cross-reactive idiotype expressed on heavy and light chains from a murine monoclonal anti-CD4.

A synthetic peptide corresponding to the second complementarity determining region (CDR2) of the immunoglobulin (Ig) variable (V) region heavy (H) chain (CDR2VH) of anti-Leu3a, a murine monoclonal antibody specific for the human CD4 molecule, was used to elicit the production of specific rabbit anti-peptide antibodies. The rabbit anti-peptide antiserum was tested for reactivity against the immunizing peptide, anti-Leu3a, and a panel of mouse monoclonal anti-CD4. Only the immunizing peptide and anti-Leu3a were recognized by ELISA, whereas the H chains of anti-Leu3a and five other monoclonal anti-CD4 preparations were recognized by Western blot analysis. These data suggest that linear structures corresponding to the CDR2VH are not normally exposed on the surface of these monoclonal antibodies and become accessible only upon unfolding of the Ig molecule. In addition, Western blot analysis demonstrated that the anti-CDR2VH peptide antiserum was able to recognize the Ig light (L) chain of anti-Leu3a. This reactivity to both H and L chains from anti-Leu3a was ascribed to a homologous five amino acid sequence region shared by the two chains. The region of homology was associated with the third framework (FR3) of the L chain and was included as a portion of the sequence in the CDR2VH synthetic peptide. This observation was confirmed by the ability of the CDR2VH anti-peptide antiserum to bind the L chains of three mouse myeloma proteins that exhibited the five amino acid sequence region of homology within their respective FR3. Together, these data provide information on the structural basis of idiotypes shared by the H and L chains from the same antibody molecule and indicate that five amino acids might be sufficient to define a minimal continuous idiotypic determinant.

Fine specificity of the murine antibody response to HIV-1 gp160 determined by synthetic peptides which define selected epitopes.

In this report, we assess the humoral immune response in inbred strains of mice immunized with baculovirus-derived recombinant HIV-1 gp160 (rgp160). Six inbred strains of mice were each immunized with two different concns (5 and 50 micrograms) of rgp160, and the antibody response to rgp160 and synthetic peptides which define distinct gp160 epitopes was examined. Within a given inbred strain of mice, no significant difference in antibody titers to gp160 was observed in those groups receiving either 5 or 50 micrograms of rgp160 per injection. Following three immunizations with rgp160, differences in anti-gp160 titers were observed among the various inbred strains; however, these differences became less apparent after additional injections with rgp160. In addition, each mouse strain exhibited a unique reactivity pattern to seven gp160 epitopes defined by synthetic peptides. Multiple injections with rpg160 were required to induce responses to certain gp160 epitopes. The observed differences in the fine specificity of the humoral immune response to distinct gp160 epitopes among the six inbred strains suggest a genetic basis for regulating the antibody response to these epitopes. This apparent regulation can be overcome by multiple injections with rgp160.