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Coral reefs are increasingly ecologically destabilized across the globe due to climate change. Behavioural plasticity in corallivore behaviour and short-term trophic ecology in response to bleaching events may influence the extent and severity of coral bleaching and subsequent recovery potential, yet our understanding of these interactions in situ remains unclear. Here, we investigated interactions between corallivory and coral bleaching during a severe high thermal event (10.3-degree heating weeks) in Belize. We found that parrotfish changed their grazing behaviour in response to bleaching by selectively avoiding bleached Orbicella spp. colonies regardless of bleaching severity or coral size. For bleached corals, we hypothesize that this short-term respite from corallivory may temporarily buffer coral energy budgets by not redirecting energetic resources to wound healing, and may therefore enable compensatory nutrient acquisition. However, colonies that had previously been heavily grazed were also more susceptible to bleaching, which is likely to increase mortality risk. Thus, short-term respite from corallivory during bleaching may not be sufficient to functionally rescue corals during prolonged bleaching. Such pairwise interactions and behavioural shifts in response to disturbance may appear small scale and short term, but have the potential to fundamentally alter ecological outcomes, especially in already-degraded ecosystems that are vulnerable and sensitive to change.

The deep ocean is the largest ecosystem on the planet, constituting greater than 90% of all habitable space. Over three-quarters of countries globally have deep ocean within their Exclusive Economic Zones. While maintaining deep-ocean function is key to ensuring planetary health, deficiencies in knowledge and governance, as well as inequitable global capacity, challenge our ability to safeguard the resilience of this vast realm, leaving the fate of the deep ocean in the hands of a few. Historically, deep-ocean scientific exploration and research have been the purview of a limited number of nations, resulting in most of humankind not knowing the deep ocean within their national jurisdiction or beyond. In this article, we highlight the inequities and need for increased deep-ocean knowledge generation, and discuss experiences in piloting an innovative project ‘My Deep Sea, My Backyard’ toward this goal. Recognizing that many deep-ocean endeavours take place in countries without deep-ocean access, this project aimed to reduce dependency on external expertise and promote local efforts in two small island developing states, Trinidad and Tobago and Kiribati, to explore their deep-sea backyards using comparatively low-cost technology while building lasting in-country capacity. We share lessons learned so future efforts can bring us closer to achieving this goal. This article is part of the theme issue ‘Nurturing resilient marine ecosystems’.

Electron microscopy of zoocin A-treated sensitive streptococcus cells revealed cytoplasmic disruption and ultimately complete rupture of the cell wall. Culture viability and optical density were shown to decrease rapidly and simultaneously in Streptococcus pyogenes FF22 but less quickly in the relatively more resistant Streptococcus mutans 10449. Zoocin A was shown to cleave hexaglycine in a colorimetric cell-free microtiter assay system, and it is concluded that the killing action of zoocin A, like that of lysostaphin, is most probably the result of direct cleavage of the peptidoglycan cross-links in the cell wall. The relationship between sensitivity to zoocin A and the peptidoglycan cross-linkage structure of Streptococcus zooepidemicus, Lactococcus spp., S. pyogenes, Streptococcus gordonii, Streptococcus oralis, S. mutans, and Streptococcus rattus has been evaluated.

SUMMARY Baculovirus-derived recombinant simian virus 40 large tumour antigen (SV40 T-ag) was used to immunize BALB/c, C57Bl/6 and CB6/F, mice and their anti-SV40 T-ag antibody responses were examined for the ability to bind synthetic peptides representing six predicted B cell epitopes on SV40 T-ag. In C57B1/6 mice, anti-SV40 T-ag antibodies failed to bind any of the six SV40 T-ag peptides. However, the antibody responses induced in both BALB/c and CB6/F| mice recognized synthetic peptides corresponding to two distinct epitopes (amino acids 690 708 and 660 679, respectively) associated with the carboxyl-terminal half of SV40 T-ag, In addition, murine MoAbs (BALB/c) generated to native SV40 T-ag, and previously characterized as recognizing the carboxyl-terminus of SV40 T-ag by deletion mutant analysis, also bound the synthetic peptide (residues 690–708) defining the carboxyl-terminus of SV40 T-ag. These data indicate that the antibody responses induced in BALB/c and CB6/r-| mice by immunization with baculovirus-derived recombinant SV40 T-ag are capable of recognizing sequential carboxyl-terminal epitopes on SV40 T-ag defined by peptides 690 708 and 660-679. respectively. No statistically significant differences in anti-SV40 T-ag antibody titres were observed between the three inbred mouse strains. These data suggested that the fine specificities of the anti-SV40 T-ag responses as assessed by synthetic peptide binding were different in the three inbred strains of mice examined. Finally, in vivo tumour challenge studies comparing recombinant SV40 T-ag with the two carboxyl-terminus peptide epitopes indicated that some tumour immunity was induced in BALB/c, but not CB6/F1 mice, by immunization with peptide 690 708 conjugated to a carrier protein. These studies suggest that the carboxyl-terminal region of SV40 T-ag represents A continuous sequential epitope involved in both the antibody response to SV40 T-ag and tumour immunity in BALB/c mice.

Nonhuman primates are often used as a model for studying vaccines for humans. However, it is not always clear how closely the antibody responses in these species mimic human responses. Recent studies have characterized the human antibody response to Haemophilus influenzae type b (Hib) in great detail. In this study, we have compared the antibody response to Hib of humans with those of other primates. Studies of isoelectric points and V kappa subgroup usage show that, like humans, nonhuman primates produce oligoclonal antibodies. Also, monkey antibodies to the Hib polysaccharide are as protective as human antibodies in an in vivo model of Hib infection. Thus, we conclude that nonhuman primates produce antibodies to Hib polysaccharide that are structurally and functionally similar to human antibodies and are a good model for testing human vaccines.

We have characterized a series of mouse monoclonal anti-CD4 and describe both their CD4 epitope recognition and Id expression. We also determined the V region gene sequences of these antibodies in an attempt to correlate epitope recognition and Id expression with V region sequence. All of these preparations recognize epitopes that cluster around the HIV gp120 binding site on the human CD4 molecule. However, we observed differences in epitope recognition among the anti-CD4 preparations, based on either competitive inhibition assays or functional assays, such as syncytium inhibition. Analysis of Id specificities using a polyclonal anti-Id generated against anti-Leu 3a indicated that five of the seven monoclonal anti-CD4 expressed a shared Id. Based on V region gene sequences, the V region kappa-chain (V[kappa]) from each of the seven antibodies was encoded by the V[kappa]21 gene family and expressed the J[kappa]4 gene segment. Those preparations that expressed the shared Id with anti-Leu 3a have virtually identical V[kappa] sequences, with a high degree of homology in the CDR. The VH region gene sequences of six of the seven antibodies also shared overall homology and appeared to be encoded by the J558 VH gene family. The seventh anti-CD4 VH region is encoded for by the VHGAM gene family. The majority of these antibodies used JH3 gene segment, although the JH2 and JH4 gene segments were also represented. In addition, several of these antibodies share a common sequence organization within their V-D-J joining regions that appears to involve N and P sequences to generate unique D segments. Together, these data suggest that differences in epitope recognition among the monoclonal anti-CD4 may reflect sequence variability primarily within the CDR3 region of both V[kappa] and VH. The basis for the detection of a shared Id most likely reflects the high degree of homology within the V[kappa] region sequences. In addition, these data, which are based on a limited analysis, suggest the possible restricted use of V region germ-line gene families in the secondary antibody response of BALB/c mice to specific epitopes on the human CD4 molecule.

A series of mouse monoclonal anti-CD4 preparations was characterized for the ability to recognize overlapping epitopes on CD4 and to inhibit HIV/simian immunodeficiency virus (SIV) syncytium formation. Based on this characterization, mAb able to recognize CD4 epitopes overlapping the HIV binding site were selected and used to immunize nonhuman primates to elicit the production of specific anti-Id antibodies. Five baboons and five rhesus monkeys were immunized with either individual or a cocktail consisting of several monoclonal anti-CD4 preparations. All the nonhuman primates produced specific anti-Id that recognized either private or cross-reactive Id depending on the monoclonal anti-CD4 used to generate the anti-Id response. Inhibition assays were performed to ascertain the ability of: 1) soluble CD4 to inhibit the Id-anti-Id reaction and 2) the various anti-Id to inhibit the CD4-monoclonal anti-CD4 reaction. These studies demonstrated that some of the anti-Id recognized a cross-reactive Id that was associated with the Ag-combining site. In addition, some of the anti-Id weakly recognized SIV gp120 by Western blot analysis. These studies may be useful in designing experiments that may lead to a better understanding of the CD4-HIV gp120 interaction and to the production of Id and/or anti-Id reagents that might be used to manipulate this virus-receptor interaction.

Mouse monoclonal anti-Id antibodies were generated against a mouse mAb (Ab-1) preparation specific for SV40 large tumor Ag (T-Ag). Four monoclonal anti-Id preparations each inhibited the binding of the monoclonal anti-SV40 T-Ag Ab-1 preparation to SV40 T-Ag. These anti-Id preparations appeared to recognize similar idiotopes on the monoclonal anti-SV40 T-Ag Ab-1 based on competitive cross-inhibition studies. One of these anti-Id preparations, designated 57B, was examined further for its in vivo modulatory capacity in mice. This anti-Id induced an Ab-3 response in BALB/c mice that recognized SV40 T-Ag (Ag+) and expressed an Id that was shared by the monoclonal anti-SV40 T-Ag Ab-1 preparation (Id+). The Id expressed on the Ab-3 differed from the Id induced in BALB/c mice immunized with the nominal SV40 T-Ag. Furthermore, characterization of the humoral immune response induced by anti-Id immunization indicated that the Ab-3 also recognized different epitopes on SV40 T-Ag when compared to the anti-SV40 T-Ag Ab-1 preparation used to generate the anti-Id. These studies indicate that monoclonal anti-Id can be used to induce humoral immune responses to a viral encoded tumor-associated Ag in vivo with 1) and Id specificity that differs from that expressed on antibodies produced by immunization with the nominal Ag and 2) an epitope specificity distinct from the Ab-1 preparation used for the production of the anti-Id.

Maternal vaccination has been proposed as a rational approach for the prevention of neonatal group B streptococcal (GBS) disease. In this study, baboons were used as a nonhuman primate model to evaluate the immunogenicity of a GBS type III glycoconjugate vaccine. Type III-specific immunoglobulin G with opsonic activity was induced after vaccination with type III polysaccharide coupled to tetanus toxoid administered with an aluminum adjuvant. This suggests that baboons could be used in evaluating maternal transfer of GBS-specific antibodies by vaccination during pregnancy.

Two plasmids encoding SV40 Tag under the control of different promoters have been examined for their ability to induce complete protection against murine experimental metastasis induced with an SV40-transformed tumour cell line. BALB/c mice immunized with a plasmid encoding SV40 Tag under the control of the SV40 promoter (pSV3neo) exhibited no detectable levels of anti-SV40 Tag antibody and were only partially protected from tumour foci development in the lungs after Intravenous tumour challenge. In contrast, mice receiving a plasmid encoding SV40 Tag under the control of the CMV promoter (pCMV-Tag) demonstrated high levels of anti-SV40 Tag antibody. These mice were completely protected from lung tumour foci development after challenge. Since antibody responses were induced only by the immunization which provided complete protection from metastatic tumour challenge, these data support the notion that antibody may play an important role in protection against experimental pulmonary metastasis within this model. Our results demonstrate that DNA immunization may serve as a possible immunotherapeutic strategy against cancers expressing tumour-specific or tumour-associated antigens.

Capsular polysaccharide production by group A Streptococcus (GAS) is controlled by transcription of the has operon that encodes the enzymes uniquely required for synthesis of the hyaluronic acid polysaccharide. To investigate the regulation of capsule gene expression during infection, we developed a reporter strain of GAS in which the has operon promoter directed transcription of green fluorescent protein (GFP). Gfp expression was triggered within minutes after introduction of the reporter strain into the peritoneal cavity of mice, as evidenced by the recovery of highly fluorescent GAS from the peritoneum 1 h after challenge. Capsule gene expression was also stimulated in the bloodstream of infected mice, as intensely fluorescent bacteria were observed in blood samples collected after either intraperitoneal or intravenous challenge. Using a similar approach, we also observed rapid induction of capsule gene expression in bacteria inoculated into the pharynx of baboons. Compared to the inoculum, increased green fluorescence was recorded in bacteria recovered from throat swabs collected 1 h after inoculation in all five animals studied. We conclude that introduction of GAS into the pharynx or into deep tissues results in rapid induction of has operon expression, a critical adaptive response that enhances GAS survival in the infected host.

Adenovirus type 19 (Ad19) infection of the human cornea results in a chronic, multifocal, subepithelial keratitis. Existing evidence suggests that early subepithelial corneal infiltrates are composed of polymorphonuclear neutrophils. In this study, the capacity of Ad19-infected human corneal stromal fibroblasts (HCFs) to produce neutrophil chemotactants (chemokines) was tested.HCFs grown from human donor corneas and passaged thrice were infected with a corneal isolate of Ad19 or mock-infected with virus-free media. Bioactivity of the cell supernatants was tested by a neutrophil chemotaxis assay. Supernatants were assayed by enzyme-linked immunosorbent assay for the neutrophil chemotactants interleukin-8 (IL-8) and GRO-alpha. Corneal facsimiles were generated with HCFs and collagen type I, infected with Ad19, and assayed by immunohistochemistry.Ad19 infection of HCFs increased neutrophil chemotaxis from a baseline of 0.4+/-0.7 cells/high-powered field (hpf; mock-infected) to 21.8+/-2.3 cells/hpf (Ad19-infected). Chemotaxis was reduced by the addition of neutralizing antibodies against IL-8 and GRO-alpha. Infection of HCFs induced quantities of IL-8 protein 300- and 1000-fold over mock-infected controls at 4 and 24 hours, respectively (33 versus 11,813 pg/mL at 4 hours, and 57 versus 76,376 pg/mL at 24 hours, Por = 0.001 for both). In contrast, GRO-alpha protein levels were only sevenfold higher at 24 hours postinfection (118 pg/mL in mock-infected controls versus 880 pg/mL in Ad19-infected cell supernatants). Neither chemokine was induced by infection of an immortalized human corneal epithelial cell line. Immunohistochemistry of infected corneal facsimiles demonstrated IL-8 in the extracellular matrix within 3 days after infection.Production of chemokines in infected tissues facilitates an early innate immune response to infection, and in the infected corneal stroma represents an elementary defense mechanism. Interleukin-8 may play a role in the development of subepithelial infiltrates in adenovirus keratitis.

Various vaccination strategies were compared for their ability to elicit antigen-specific tumour immunity, using the SV40-BALB/c murine tumour system. Specifically, mice were injected with baculovirus-derived recombinant SV40 Tag (rTag), synthetic peptides corresponding to B cell epitopes on SV40 Tag or a plasmid DNA construct encoding the gene for SV40 Tag. In vivo tumour immunity was determined by a lethal tumour challenge with syngeneic SV40-transformed tumour cells. SV40 Tag-specific antibody titres were induced in mice immunized with rTag or Tag synthetic peptides. Partial tumour protection was observed in mice that were immunized with SV40 Tag peptides, where as complete tumour immunity was observed in mice immunized with rTag. Although protective tumour immunity was also observed in mice immunized with DNA, negligible levels of antibodies to SV40 Tag were detected. Examination of the cytotoxic T lymphocyte (CTL) activity in mice injected with the SV40 Tag-DNA construct revealed Tag-specific lysis of syngeneic SV40-transformed tumour cells. Conversely, little to no CTL activity was detected in mice immunized with rTag. However, antigen-specific antibodies from rTag immunized mice were capable of mediating antibody-dependent cell-mediated cytotoxicity against SV40-transformed cells. These data indicate that the immune mechanisms elicited for protection against SV40 induced tumours in mice appeared to be dependent on the vaccination strategy employed and included both humoral and cell-mediated immune responses.

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.

Ocular surface immune mechanisms are subservient to the fine function of the eye. A clear cornea with a smooth, well-lubricated facade is prerequisite to lucid vision. Hence, corneal inflammation and post-inflammatory scarring are intolerable, and the cornea contains a minimum of lymphoid elements. Although conjunctival dysfunction and consequent tear film deficiency can malign the corneal surface, conjunctival inflammation is tolerated to a considerable degree. In contrast to the human cornea, human conjunctiva contains an abundance of lymphoid tissue. Certain aspects of human conjunctival immunology elicit little debate. Langerhans cells are abundant in conjunctival epithelium. Isolated CD8+ suppressor/cytotoxic T cells predominate in conjunctival epithelium, while T cells in the substantia propria distribute equally between CD4+ T helper cells and CD8+ cells. Yet the presence of plasma cells in human conjunctiva, the expression of secretory component by human conjunctival epithelium, and the function of human conjunctival lymphoid follicles are in dispute. Confusion may derive in part from the use of inappropriate animal models; rodent conjunctiva does not appear to be a worthy facsimile for human conjunctiva. Discrepancies between different human studies likely result from variance in subject age, biopsy site and extent, histologic or histochemical technique, and perhaps the degree of inflammation present at the time of biopsy. Careful immunohistochemical and in situ molecular assays on well-defined loci within the conjunctiva of comparable human subjects may resolve such questions in the future. Organized mucosa-associated lymphoid tissue is rigorously defined as mucosal lymphoid follicles with an ultrastructurally distinct overlying lymphoepithelium. Based on available evidence, the epithelium overlying mammalian conjunctival lymphoid follicles does not contain distinct M cells. Whether zonal differences in morphology reflect real differences in the capacity to sample tear film antigens for presentation to the mucosal immune system remains to be established.

A wide variety of immunotherapeutic strategies for the treatment of cancers is currently under investigation in both animal models and in clinical human trials (reviewed in Dranoff and Mulligan 1995). These include in vitro expansion and adoptive transfer of tumor-specific cytotoxic T cells in the presence or absence of recombinant cytokines (Rosenberg et al. 1985, 1986, 1988, 1989, 1991, 1993; Teng et al. 1991), tumor antigen-based vaccines (Cheever et al. 1986; Bright et al. 1994a; Conry et al. 1994; Stevenson et al. 1995), and tumor-reactive monoclonal antibodies conjugated to toxins (Trail et al. 1993; Vivetta et al. 1993). One of the most recent and innovative strategies has been based on the concept of genetic immunization. It is now well established that vaccination with plasmid DNA encoding a specific gene can elicit strong, long-lived protective immune responses to a variety of infectious agents (reviewed in Ulmer et al. 1996; Whalen 1996) as well as certain tumors (Conry et al. 1994; Nabel et al. 1994b; Stevenson et al. 1995; Bright et al. 1996; Corr et al. 1996; Schirmbeck et al. 1996). DNA vaccines directed at eliciting an immune response toward tumors appear to offer promise for both the prophylactic and therapeutic treatment of cancer.

Spermatogenesis is a complex differentiation process in which diverse stage-specific proteins are co-ordinately expressed. Previously, subtractive hybridization and differential hybridization have been used in the identification of differentially expressed mRNAs. Although these techniques have been successfully used they require large amounts of RNA and are time consuming. To overcome these problems we have made use of the recently described mRNA differential display technique. The technique is an effective method which can identify and separate cDNAs that are differentially expressed between various cell-types. By comparing RNA from testes of mature (.60 days old) and prepubertal (15‐16 days old) mice we have identified nine differential cDNA bands expressed in mature testes. The differential display cDNA band DDC8 was used to screen a testis cDNA library and the full length cDNA was isolated and sequenced. DDC8 cDNA is 1965 bp with an open reading frame of 533 amino acids which codes for a predicted hydrophilic protein with a calculated molecular weight of 62.04 kDa. RNase protection assays indicate DDC8 to be expressed during the postmeiotic stages of spermatogenesis and database searches using both nucleotide and amino acid sequences show DDC8 to have similarities to structural, cytoskeletal and associated proteins.

A study was undertaken to evaluate the effect on nuclei number in human embryos cultured in vitro with primary cell lines of human Fallopian tube epithelium. The development of 203 surplus human embryos, cultured in standard culture medium (Earle's balanced salt solution + 15% A5) with or without ampullary cells, was observed from day 2 to day 5.5 post-insemination. The expanded blastocysts in both culture conditions were analysed for nuclei numbers per blastocyst. Embryos transferred to co-culture at the 2-cell stage had an average of 120.7 nuclei per blastocyst, which was significantly higher than the average of 62.9 nuclei per blastocyst (P = 0.023) for the embryos transferred to co-culture at the 4-cell stage. The embryos cultured in the control medium had an average of 42.1 nuclei per blastocyst, which was significantly less than co-cultured embryos (P = 0.04). Severely fragmented embryos (grades 3 and 4) did not show recovery in co-culture. Our results show that when human embryos are transferred to co-cultures before the 4-cell stage, the blastulation rate and the cell number per embryo increase significantly compared to the embryos cultured in standard culture medium. The possible effect of co-culture on embryonic gene expression is discussed.

Increasingly strong medical and political pressures are stimulating consideration of the transplantation of baboon organs and cells into humans. Critical to the success of these xenotransplants is management of the immune system such that graft rejection and, in the case of bone marrow transplantation, graft-versus-host disease do not result in transplant failure. The polymorphic products of the major histocompatibility complex (MHC) are the primary barrier to successful allotransplantation, and here we describe class I MHC molecules from baboon (Papio anubis) to gain an understanding of how similarities and differences between baboon and human MHC molecules might affect xenograft survival and function. Comparative analyses of our five novel baboon class I molecules with defined HLA class I molecules demonstrate that the baboon class I molecule are up to 90% identical. Disparity between baboon class I proteins and their human homologues lies predominately at positions in the antigen-binding groove, while C-terminal portions of the class I heavy chain are more conserved between the two species. Such concentration of cross-species differences within the alpha1 and alpha2 domains involves a majority of substitutions at positions demonstrating polymorphism in human alleles; the location of substitutions distinguishing baboon and human molecules thus resembles the positioning of human class I allopolymorphisms. Because this preliminary characterization indicates that both baboon and human T cells with be restricted by xenogeneic class I molecules, immune responses triggered during baboon-to-human transplantation should mimic those arising during MHC mismatched human allotransplantation.

Rochalimaea (Bartonella) henselae is a fastidious, slowly growing, gram-negative bacillus that is an etiologic agent of bacillary angiomatosis, cat scratch disease, and related syndromes. Accumulation of direct microbiologic evidence of the relationship between the organism and the syndromes compatible with cat scratch disease has been hindered by the difficulties in the primary isolation of the organism from infected tissue specimens. A chemically defined liquid medium was developed to support the growth of Rochalimaea species to facilitate study of the organism. This medium was also used successfully to isolate R. henselae from clinical specimens from infected patients and a domestic cat. Recovery of R. henselae in this was more successful than when recovery was attempted on solid agar. This cell-free, extract-free, defined medium additionally supported the growth of Rochalimaea quintana and Afipia felis.

In this report, we describe an immune mechanism that seems to play a role in tumor immunity in BALB/c mice challenged with SV40-transformed cells. This immune mechanism was induced by immunization of mice with rSV40 large tumor Ag (T-Ag). After rSV40 T-Ag immunization, BALB/c mice were protected from a lethal tumor challenge with syngeneic SV40-transformed cells (mKSA). Specifically, we examined CTL activity, NK activity, complement-dependent cytotoxicity, and Ab-dependent cell-mediated cytotoxicity (ADCC) in rSV40 T-Ag-immunized mice that were protected from a subsequent lethal tumor challenge. Immune splenocytes from surviving animals that were subsequently primed in vitro with rSV40 T-Ag demonstrated little to no SV40 T-Ag-specific CTL activity. In addition, natural immunity, as assessed by NK lytic activity, was comparable to that of unimmunized animals. Similarly, minimal significant complement-dependent cytotoxicity of radiolabeled mKSA cells was demonstrated by SV40 T-Ag-specific serum Abs. However, SV40 T-Ag-specific Abs using peritoneal exudate cells as effectors, exhibited significant levels of ADCC against radiolabeled mKSA target cells. Together, these studies indicate that rSV40 T-Ag immunization elicited ADCC that may represent a potential humoral immune mechanism involved in the protection of BALB/c mice from a lethal challenge with syngeneic SV40-transformed cells.

Borrelia burgdorferi, an agent of Lyme disease, is killed by some monoclonal antibodies in the absence of complement or phagocytes. In the present study, the bactericidal action of monoclonal antibodies against B. burgdorferi and B. hermsii, a cause of relapsing fever, was further characterized. H6831, an antibody recognizing the OspB proteins of some B. burgdorferi strains, and H4825, an antibody specific for one serotype of B. hermsii, were purified, and Fab fragments of the antibodies were prepared. In time-kill studies, more than 99.9% of strain B31 B. burgdorferi cells were killed after 30 min of exposure to H6831 Fab fragments. The MBC of the Fab fragments was 10 micrograms/ml. Electron microscopy revealed that the bactericidal Fab fragments produced numerous blebs and cell lysis of the borrelias for which they were specific. To identify the epitope for H6831, the OspB sequences of H6831-susceptible and -resistant strains and mutants were determined. The deduced OspB proteins of all H6831-resistant strains and mutants differed from the strain B31 OspB at residue 253. Murine antisera raised against a 21-mer synthetic peptide representing the region around residue 253 were specific for strain B31 by Western blot (immunoblot) and growth inhibition assays. Furthermore, the antipeptide serum inhibited the binding of H6831 to whole borrelias. These findings indicated that the linear component of the bactericidal antibody's epitope was located at or near residue 253.

The reversible denaturant-induced unfolding of immunoglobulin molecules has been analyzed by transverse urea gradient gel electrophoresis and the effects that urea-induced unfolding exerts on the functional properties associated with their variable region, i.e. antigen binding and idiotypic expression, have been determined by Western blot analysis. Results obtained from these experiments indicate that urea-induced unfolding of the immunoglobulin molecule is a highly cooperative reversible process that occurs through a two-state transition with no accumulation of intermediates. The unfolding transition has its midpoint at about 6.5 M urea and appears to be slow on the time scale of electrophoresis. Folding intermediates in rapid equilibrium with the unfolded state as well as molecular forms with different electrophoretic mobility can be detected during refolding reactions. Results from Western blot analysis confirm the highly cooperative reversible urea-induced unfolding of immunoglobulin molecules and demonstrate that the unfolding transition leads to disappearance of both antigen binding and idiotypic expression, whereas the ability to interact with antibodies directed to continuous epitopes of the variable region is preserved. After progressive removal of the denaturing agent, the variable region refolds into structures that regain the functional properties of the native conformation.

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.

The third variable region (V3) of the HIV-1 gp120 envelope molecule appears to represent a target for naturally occurring neutralizing antibodies in HIV-1-infected individuals. In this report, we examined the extent of antibody cross-reactivity to a panel of V3-based synthetic peptides in six inbred strains of mice following repeated immunization with a baculovirus-derived recombinant gp160 (rgp160) preparation formulated with alum. The amino acid sequence of the rgp160 used in these immunizations was based upon the HIV-1 IIIB (LAI) isolate. Following five injections with rgp160, all six strains developed antibodies to the homologous IIIB-based V3 peptides, designated 304-321 and RP135. However, antibody cross-reactivity to the other nonhomologous V3 peptides was either undetectable or limited among the strains of mice examined. No in vitro neutralizing activity against HIV-1 was observed in sera from any of the six inbred strains of mice that were examined. These results suggest that repeated immunization of mouse strains with a rgp160/alum formulation leads to nonneutralizing antibodies directed against the V3 region which remain predominantly type specific.

A panel of mouse monoclonal anti-CD4 antibodies was characterized in terms of idiotypic expression by using specific anti-idiotypic antibody (anti-Id) reagents generated in rabbits immunized with anti-Leu3a, a monoclonal anti-CD4 which inhibits the human immunodeficiency virus (HIV) gp120 binding to CD4. Direct binding and competitive inhibition assays demonstrate that the majority of monoclonal anti-CD4 antibodies able to recognize CD4 epitopes overlapping the epitope recognized by anti-Leu3a expressed an antigen-combining site-related cross-reactive idiotype (IdX). Western blot analysis was used to demonstrate that this IdX is associated primarily with the light (L) chain of the monoclonal anti-CD4 antibodies. To further characterize the structural basis of the IdX, the nucleotide sequence of the variable region of the L kappa chain of anti-Leu3a was determined. Peptides corresponding to the first, second, and third complementarity determining regions (CDRs) of the L chain of anti-Leu3a were synthesized and used to immunize rabbits. All anti-peptide antisera recognized the immunizing peptide, the cognate anti-Leu3a molecule, and several other monoclonal anti-CD4 antibodies by direct binding assays. Western blot analysis utilizing the anti-CDR peptide reagents demonstrates that the reactivity to the monoclonal anti-CD4 antibodies was L chain-specific. The anti-Id generated by immunizing with the intact anti-Leu3a molecule failed to recognize the three L chain-derived CDR synthetic peptides, suggesting that the IdX requires the presence of the three-dimensional configuration of the L chain for its expression. The broad range of reactivity exhibited by the antipeptide antisera indicates that the majority of mouse monoclonal anti-CD4 antibodies characterized in this study utilize L chains encoded by a single germ line variable (V) region kappa (V kappa) chain gene or by V kappa genes that belong to the same gene family.

A synthetic peptide corresponding to the third complementarity determining region (CDR) of the heavy chain (CDR3VH) of anti-Leu3a, a monoclonal anti-CD4 antibody which inhibits HIV gp120 binding to CD4, was used to elicit specific anti-peptide antibodies in rabbits. The anti-peptide antisera showed anti-idiotypic antibody (anti-Id) activity and recognized both the immunizing peptide and the intact cognate protein by ELISA. In addition, the antisera reacted with isolated heavy chains of anti-Leu3a by Western blot analysis. The lack of reactivity with a panel of monoclonal anti-CD4 antibodies suggested that the anti-peptide antisera recognize a private idiotype (Id) associated with the anti-Leu3a CDR3VH region. Further studies demonstrated the inability of the rabbit antisera to inhibit the binding of anti-Leu3a to the CD4 molecule. In addition, soluble recombinant CD4 was unable to inhibit the binding of the rabbit anti-peptide antisera to anti-Leu3a indicating that the CDR3VH region may not be involved in CD4 recognition. Anti-Id containing sera from mice, rabbits and nonhuman primates immunized with the intact anti-Leu3a molecule did not bind the CDR3VH synthetic peptide, suggesting that the corresponding region of anti-Leu3a may not represent an immunodominant idiotypic determinant in thes e species. These results suggest the potential use of synthetic peptides corresponding to immunoglobulin variable (V) region amino acid sequences in generating anti-Id reagents of a predefined specificity. In addition, V-region synthetic peptides may be useful in mapping the idiotopes recognized by an anti-Id response to the cognate molecule.

Idiotypes (Id) are antigenic determinants expressed on the variable (V) region of the immunoglobulin molecule. Id-bearing antibodies, or Ab-1, are produced upon stimulation with a given antigen. Ab-1 may elicit the production of anti-idiotypic antibodies (anti-Id) or Ab-2. The anti-Id also expresses Id determinants and may in turn elicit the production of anti-anti-Id or Ab-3. The production of Ab-1, Ab-2, and Ab-3 responses resulting from stimulation with the antigen is representative of components within an Id cascade. The existence of this Id cascade is the basis for the development of Id based strategies for controlling the immune response to infectious agents and tumors. In this review we will focus on several aspects regarding the Id cascades that may be operational during the immune response to the human immunodeficiency virus (HIV). In light of several studies which suggest the existence of Id-anti-Id interactions operating during the course of HIV infection, we will discuss the potential applications of Id based strategies in manipulating the immune response to HIV.

Purpose An experimental pulmonary metastatic model for ovarian cancer has still not been developed. Here we introduce tumor models in NOD/SCID mice for ovarian cancer incorporating a tumor-specific Sp17/CT antigen. Methods Ovarian adenocarcinoma cells (SKOV-3) were intraperitoneally injected in an attempt to demonstrate a seeding of the organs located in the peritoneal cavity. Examination of the ovaries for the progression of the disease was marked by an increase of the size of the growing tumor. Following intravenous injection, viable SKOV-3 cells were recovered by primary organ cell culture in the liver, lungs, ovaries, abdomen, and ascites. Summary The presence of SKOV-3 cells in these tissues was confirmed by RT-PCR and immunocytochemistry using the sperm protein 17 (Sp17). The number and size of tumoral foci were quantified using a computer-aided model. Tumoral foci were observed in the lungs of tumor-bearing mice and they are dependent on the dose of injected SKOV-3 cells and time after injection. Conclusion The quantification of the number of lung tumoral foci and the detection of distant metastasis might provide specific reference points for examining the mechanism(s) of the immune response to Sp17 and for evaluating immunologically based therapies within this novel ovarian murine tumor model.

The sera of 57 patients with active, untreated pulmonary tuberculosis were examined for the presence of a common anti-DNA idiotype, 16/6. Thirty-four of the 57 sera (60%) had an increased level of the idiotype, as measured by enzyme-linked immunosorbent assay using a specific rabbit anti-16/6 serum. Of 28 matched control sera, only 1 (4%) was found to be positive for the idiotype. The sera of patients with tuberculosis also showed increased activity against a variety of antigens with which lupus autoantibodies are known to cross-react (e.g., single-stranded DNA, double-stranded DNA, polynucleotides, and cardiolipin). A correlation was observed between serum IgG and IgM levels and the 16/6 idiotype levels.

Serum neutralizing antibodies against the human immunodeficiency virus were frequently detected in infected individuals, and low or absent serum neutralizing titers correlated with poor prognosis. Multiple diverse human immunodeficiency virus isolates were found to exhibit similar susceptibility to neutralization by a panel of human seropositive sera, suggesting that neutralizing antibodies are largely directed against conserved viral domains. Furthermore, utilizing antisera raised against a library of synthetic env peptides, four regions which are important in the neutralization process have been identified within both human immunodeficiency virus envelope glycoproteins (gp41 and gp120). Three of these are in conserved domains and should be considered for inclusion in a candidate vaccine.

Anti-idiotype antibodies to a mouse monoclonal antibody A-12 directed against HBsAg were produced in rabbits. The anti-Id consisted of an Ab-2 alpha preparation that did not display any detectable internal image activity. Immunization of BALB/c mice with the anti-Id (Ab-2 alpha) coupled to KLH induced an anti-HBs response without subsequent HBsAg exposure. No anti-HBs was detected in control groups of mice immunized with other rabbit anti-Id-KLH preparations. The anti-HBs containing sera from mice immunized with the Ab-2 alpha were able to inhibit the Id-anti-Id reaction, indicating that an Id-positive, anti-HBs response was induced. This idiotype is not normally expressed during the murine immune response to HBsAg and suggests that noninternal image anti-Id activates silent clones. This study, along with our previous results obtained with the use of internal image anti-Id, suggests that there is more than one Id network operational during the BALB/c murine immune response to HBsAg.

Two IgM monoclonal antibodies (McAb) that recognized distinct antigenic determinants of herpes simplex virus type 2 (HSV2) were shown to share idiotypes. Seven other IgM McAb generated to either HSV2 or the major envelope glycoprotein of HSV2, VP119, did not express this shared idiotype. The idiotypic determinants were associated with the antibody-combining site, because HSV2 inhibited the idiotype-anti-idiotype reactions. Anti-HSV2 sera raised in BALB/c mice also expressed the idiotype shared by the two anti-HSV2 McAb.