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B Golding

Publications and source records attributed to B Golding.

At least 19 recordsLinked to original sources
PubMed1998

HIV peptide conjugated to heat-killed bacteria promotes antiviral responses in immunodeficient mice.

Enhancement of immunity in the setting of HIV infection is difficult owing to loss of functional CD4+ T cells. The MHC class II-deficient mouse (II-/-) environment simulates that of the immunocompromised HIV-infected individual, since these mice have low CD4+ T cell numbers, defective CD4-dependent responses, and are susceptible to opportunistic infection. This strain was used to test whether heat-killed Brucella abortus (BA), covalently conjugated to the V3 peptide of HIV-1 (MN), could elicit anti-HIV responses. V3-BA, but not the T-dependent antigen V3-KLH, induced high levels of IL-12, IFN-gamma, and IL-10 mRNA in both wild-type (WT) and II-/- mice within 24 hr of injection. V3-BA-treated, but not V3-KLH-treated, II-/- mice developed serum IgG and IgA anti-V3 antibodies, with IgG2b and IgG3 as the predominant isotype. Viral neutralization studies, using a syncytium inhibition assay, demonstrated that the antibodies generated by V3-BA in II-/- mice were capable of neutralizing HIV. These experiments demonstrate that a heat-inactivated bacterium such as BA, when used as a carrier, can generate a cytokine environment that results in the production of neutralizing antiviral antibodies in an immunodeficient host. Such strategies could be important in the development of immunotherapies and vaccines for HIV-1 patients.

AIDS Vaccines↗
PubMed1998

Induction of HIV-1 IIIb neutralizing antibodies in BALB/c mice by a chimaeric peptide consisting of a T-helper cell epitope of Semliki Forest virus and a B-cell epitope of HIV.

A colinearly synthesized peptide consisting of a H-2d restricted T-helper cell epitope of Semliki Forest virus (SFV) and triple repeats of sequence GPGRAF, derived from the V3 domain of HIV-1 strains, was used to immunize BALB/c (H-2d) mice. Pepscan analysis of sera from peptide-immunized mice revealed that the chimaeric peptide GREKFTIRPHYGKEIGPGRAFGPGRAFGPGRAF contains three distinct antibody-reactive sequences GREKFTIR, PHYGKEI and GPGRAF. The chimaeric peptide evoked HIV-1 IIIb neutralizing antibodies in serum as measured in vitro by reduction of syncytia formation and reduction of p24 production as well. So, the T-helper cell epitope of SFV provided help to a small linear neutralization epitope of HIV-1 strains. Interestingly, the T-helper cell epitope alone might induce antibodies cross-reactive with HIV-1 IIIb specific peptide GPGRAFVTIGK which shows some homology (residues underlined) with the antibody-reactive sequence GREKTIR of SFV.

Amino Acid Sequence↗
PubMed1997

Inhibition of primary and recall allergen-specific T helper cell type 2-mediated responses by a T helper cell type 1 stimulus.

Allergic responses are characterized by the production of Ag-specific IgE Abs that are dependent upon Th2-mediated T cell help. We determined whether heat-killed Brucella abortus (BA), an inducer of Th1 responses, could influence the allergic Th2-mediated IgE response to OVA adsorbed to alum (O/A). BA plus O/A, but not O/A alone, induced high levels of mRNA for IFN-gamma and IL-12 promptly after injection. Furthermore, initial treatment with BA plus O/A rendered both BALB/c and C57Bl/6 mice incapable of mounting high IgE responses even after repeated challenges with allergen alone. Long term abrogation of anti-OVA IgE correlated with an increased frequency of IFN-gamma-secreting OVA-specific cells and a decreased frequency of IL-4-secreting OVA-specific cells. Initial treatment with anti-IL-12 prevented BA-induced early IFN-gamma production and secondary IgG2a responses, but did not abrogate IgE suppression. Additionally, secondary OVA-specific IgE responses were down-regulated by BA conjugated to OVA or by BA given with O/A. BA-induced down-regulation of secondary IgE responses was associated with increased frequency of Ag-specific IFN-gamma-secreting cells. These results suggest the possibility that even recall Th2-mediated immune responses can be attenuated if Ag is given with a carrier or adjuvant that induces potent Th1-promoting cytokines.

Allergens↗
PubMed1997

Phylogenetic analysis of the Caliciviruses.

A phylogenetic portrait of the genus Calicivirus in the family Caliciviridae was developed based upon published sequences and newly characterized calicivirus (CV) strains, including additional Sapporo-like HuCV strains in pediatric diarrhea stool specimens from South Africa, the United Kingdom, and the United States. Distance and parsimony methods were applied to nucleotide and amino acid sequences of human and animal calicivirus 3D RNA-dependent RNA polymerase (approximately 470nt) and capsid hypervariable regions (approximately 1,200nt) to generate phylogenetic trees. Pairwise amino acid identity in the 3D region among the Sapporo-like strains ranged from 61% to 100%. Human and animal caliciviruses (HuCVs and AnCVs) separated into five genogroups: small round-structured viruses (SRSV), Sapporo-like, and hepatitis E virus (HEV)-like HuCVs and rabbit-, and vesicular exanthema of swine virus (VESV)-like AnCVs, each with a distinct genome organization. Each genogroup, including the Sapporo-like HuCVs, subdivided further into subgenogroups. The capsid region trees had higher levels of confidence than the 3D region trees and limited conclusions about genogroups could be drawn from the 3D region analyses. This analysis suggested that CVs include five potential virus subfamilies.

Amino Acid Sequence↗
PubMed1996

Evolution: when was life's first branch point?

A recent analysis of protein sequences from diverse organisms has estimated that all extant species share a common ancestor that lived only 2 billion years ago; but how can this be squared with the fossil evidence that complex cells existed up to 3.5 billion years ago?

Animals↗
PubMed1996

Partial characterization of the genome of nine animal caliciviruses.

Caliciviruses (CVs) include at least 42 distinct serotypes. Seventeen CV serotypes have been isolated from marine sources and are called San Miguel sea lion caliciviruses (SMSVs). CVs also have been isolated from reptiles, primates, and other terrestrial animals. Nucleotide sequences from portions of genome of prototype strains for six SMSV serotypes, the reptile CV, Cro-1, the cetacean CV, Tur-1, and the primate CV, Pan-1, are presented. cDNA products of the polymerase (all strains characterized) and capsid (SMSV-17) regions were produced by reverse transcription-polymerase chain reaction using Pan-1 primers. Comparisons of nucleotide and amino acid identity among these and published CV sequences indicated that the nine characterized CVs fall into a phylogenetic group that includes SMSV-1 and SMSV-4 and that is more closely related to other characterized animal CVs than to most human CVs. The phylogenetic analysis also indicated that distinct genera exist among the Caliciviridae. SMSV-17 and SMSV-4 are predicted to be closer to each other than other caliciviruses of known serotype; 574 (82%) of the 704 amino acids in the SMSV-17 and SMSV-4 capsid genes were identical.

Amino Acid Sequence↗
PubMed1996

Brucella abortus as a potential vaccine candidate: induction of interleukin-12 secretion and enhanced B7.1 and B7.2 and intercellular adhesion molecule 1 surface expression in elutriated human monocytes stimulated by heat-inactivated B. abortus.

Development of a vaccine which is capable of generating a strong cellular immune response associated with gamma interferon (IFN-gamma) production and cytotoxic T-cell development requires that the immunogen be capable of inducing the secretion of interleukin-12 (IL-12), which is a pivotal factor for the differentiation of Th1 or Tc1 cells. We have previously shown that the heat-inactivated gram-negative bacterium Brucella abortus can induce IFN-gamma secretion by T cells. In the present study, we demonstrate that B. abortus and lipopolysaccharide (LPS) from B. abortus can induce IL-12 p40 mRNA expression and protein secretion by human elutriated monocytes (99% pure). p40 mRNA was detected within 4 h, and p40 protein could be measured at 24 h. This induction was abrogated by anti-CD14 monoclonal antibody, suggesting that monocytes recognize B. abortus via their receptor for LPS. The biological activity of IL-12 secreted by B. abortus-stimulated monocytes was demonstrated by its ability to upregulate IFN-gamma mRNA expression in T cells separated from monocytes and B. abortus by a transwell membrane. The B. abortus-induced IL-12 also enhanced NK cytolytic activity against K562 target cells. B. abortus was shown to rapidly increase the expression of the costimulatory molecules B7.1 and B7.2 and intercellular adhesion molecule 1 on human monocytes. Together, these data indicate that B. abortus can directly activate human monocytes and provide the cytokine milieu which would direct the immune response towards Th1-Tc1 differentiation.

Antigens, CD↗
PubMed1996

Brucella abortus conjugated with a peptide derived from the V3 loop of human immunodeficiency virus (HIV) type 1 induces HIV-specific cytotoxic T-cell responses in normal and in CD4+ cell-depleted BALB/c mice.

We have previously shown that immunization of mice with human immunodeficiency virus (HIV)-derived proteins or peptides conjugated to inactivated Brucella abortus induces the secretion of virus-neutralizing antibodies, predominantly of the immunoglobulin G2a (IgG2a) isotype. In addition, B. abortus activates human CD4+ and CD8+ cells to secrete gamma interferon. Since these are both characteristics of a Th1-type immune response, which is associated with the development of cell-mediated immunity, it was important to determine if B. abortus conjugates would also act as a carrier to induce a cytotoxic T-lymphocyte (CTL) response. To test this hypothesis, we conjugated an 18-amino-acid peptide from the V3 loop of the MN strain of HIV-1 gp120 that contains both B- and cytotoxic T-cell epitopes to B. abortus (B. abortus-MN 18-mer). A 10-amino-acid fragment of this peptide has been shown to be the minimal CTL determinant presented by murine H-2Dd. It was found that two in vivo immunizations with 10(8) organisms of B. abortus-MN 18-mer followed by in vitro stimulation with peptide induced a virus-specific CTL response. Conjugation to B. abortus was required for in vivo priming, since there was no induction of memory CTLs when B. abortus was only mixed with peptide. Targets pulsed with peptide as well as those infected with a vaccinia virus encoding HIV gp160 were killed, demonstrating recognition of naturally processed envelope. Also, major histocompatibility complex-incompatible L cells which were infected with vaccinia viruses that encoded H-2Dd, but not H-2Kd, and pulsed with peptide were lysed. This demonstrated the appropriate major histocompatibility complex class I restriction. Treatment of the mice with anti-L3T4 prior to immunization caused a severe depletion of CD4+ lymphocytes, yet it did not decrease the CTL priming. Thus, inactivated B. abortus can induce non-CD4+ cells to produce the cytokines required for CTL induction. We conclude that B. abortus stimulates a cellular as well as a humoral immune response, even in the relative absence of CD4+ helper cells. It may be a particularly useful vaccine carrier in HIV-1-infected individuals or others with impaired CD4+ T-cell function.

AIDS Vaccines↗
PubMed1995

Vaccine strategies: targeting helper T cell responses.

Vaccine strategies need to take into account the balance of T helper subsets they induce. TH1 cells, which secrete IFN gamma and IL-2, are associated with CMI, rather than humoral responses, and afford protection against intracellular infections including parasites. In contrast, TH2 cells secrete IL-4, IL-5, and IL-10; elicit high-titer antibody responses and poor CMI; and are associated with susceptibility to infection with intracellular pathogens. Depending on the type of TH cell bias required, it is possible to manipulate the immune response to a protein or peptide by employing (1) different adjuvants, (2) conjugating the protein to various carriers, (3) immunizing in the presence of cytokines, (4) using alternative routes of administration, or (5) using different forms or doses of antigen. To apply these approaches to a particular vaccine, it is necessary to identify which component of the infectious agent (e.g., envelope protein or peptide) or allergen to target. Once the type of TH cell response that is protective is identified, it may be possible to combine a protein with an adjuvant or link it to a carrier that will promote responses towards the most advantageous TH subset.

Adjuvants, Immunologic↗
PubMed1995

Monophosphoryl lipid A behaves as a T-cell-independent type 1 carrier for hapten-specific antibody responses in mice.

It is known that the lipopolysaccharide (LPS) of gram-negative bacteria, in addition to being a potent adjuvant, is an effective carrier for covalently associated haptens. However, the toxic nature of most forms of LPS precludes their use as adjuvants or carriers for human vaccines. 4'-Monophosphoryl lipid A (MLA), a derivative of LPS with attenuated toxicity, is currently being tested in humans as an immunological adjuvant. In this study, MLA was tested for its ability to function as a carrier for a small hapten, the trinitrophenyl group (TNP). MLA was first modified by addition of 6-aminocaproic acid to the 6' position of the disaccharide backbone (Cap-MLA). TNP was then attached to Cap-MLA via the free amino group, yielding TNP-Cap-MLA. Immunization of normal mice with TNP-Cap-MLA resulted in high-titer anti-TNP responses of immunoglobulin M and all immunoglobulin G subclasses. Furthermore MLA, like other T-cell-independent type 1 (TI-1) carriers, induced responses in athymic and X-linked immunodeficient mice. In all cases, immunization with either MLA alone or TNP-Cap plus MLA failed to induce measurable anti-TNP antibodies of any isotype, indicating that covalent association of MLA and hapten was necessary for MLA's carrier activity to be manifested. These properties of MLA make it a potential candidate as a carrier for vaccine subunit components, such as small peptides, especially for situations in which T-cell help is impaired, as occurs following human immunodeficiency virus type 1 infection.

Animals↗
PubMed1995

Human peripheral blood CD4+ and CD8+ T cells express Th1-like cytokine mRNA and proteins following in vitro stimulation with heat-inactivated Brucella abortus.

Defining the pattern of lymphokine production associated with Brucella abortus is critical for advancing the development of B. abortus as a vaccine carrier. In the present study we investigated the ability of heat-inactivated B. abortus or lipopolysaccharide from B. abortus to induce lymphokine production from purified human T cells in vitro. Gamma interferon (IFN-gamma), interleukin-2 (IL-2), IL-4, and IL-5 induction was assayed by mRNA-specific PCR and by enzyme-linked immunosorbent assay and bioassay for protein production. Following depletion of monocytes and B cells, B. abortus increased IFN-gamma and IL-2 mRNA expression in purified T cells compared with expression in unstimulated cells. In contrast, no IL-5 mRNA expression and only transient low-level IL-4 mRNA expression and no IL-4 protein secretion were detected. Phytohemagglutinin or phorbol myristate acetate plus ionomycin induced mRNA and protein for all these cytokines. Similar results were obtained with LPS purified from B. abortus. Removal of NK cells did not reduce lymphokine production, and enriched NK cells did not express IFN-gamma mRNA or secrete IFN-gamma protein in response to B. abortus, indicating that NK cells were not the responding population. Both CD4+ and CD8+ populations produced IFN-gamma and IL-2 in response to B. abortus. Preincubation of resting T cells with B. abortus or LPS from B. abortus for 7 days induced their differentiation into Th1-like cells as judged by their subsequent lymphokine response to phorbol myristate acetate plus ionomycin. These results suggest that B. abortus can induce differentiation of Th0 into Th1-type cells.

Base Sequence↗
PubMed1995

Brucella abortus conjugated with a gp120 or V3 loop peptide derived from human immunodeficiency virus (HIV) type 1 induces neutralizing anti-HIV antibodies, and the V3-B. abortus conjugate is effective even after CD4+ T-cell depletion.

Human immunodeficiency virus type 1 (HIV-1) infection is associated with loss of function and numbers of CD4+ T-helper cells. In order to bypass the requirement for CD4+ cells in antibody responses, we have utilized heat-inactivated Brucella abortus as a carrier. In this study we coupled a 14-mer V3 loop peptide (V3), which is homologous to 9 of 11 amino acids from the V3 loop of HIV-1 MN, and gp120 from HIV-1 SF2 to B. abortus [gp120(SF2)-B. abortus]. Our results showed that specific antibody responses, dominated by immunoglobulin G2a in BALB/c mice, were induced by these conjugates. Sera from the immunized mice bound native gp120 expressed on the surfaces of cells infected with a recombinant vaccinia virus gp160 vector (VPE16). Sera from mice immunized with gp120(SF2)-B. abortus inhibited binding of soluble CD4 to gp120, whereas sera from mice immunized with V3-B. abortus were ineffective. Sera from mice immunized with either conjugate were capable of blocking syncytium formation between CD4+ CEM cells and H9 cells chronically infected with the homologous virus. Sera from mice immunized with gp120(SF2)-B. abortus were more potent than sera from mice immunized with V3-B. abortus in inhibiting syncytia from heterologous HIV-1 laboratory strains. Importantly, in primary and secondary responses, V3-B. abortus evoked anti-HIV MN antibodies in mice depleted of CD4+ cells, and sera from these mice were able to inhibit syncytia. These findings indicate that B. abortus can provide carrier function for peptides and proteins from HIV-1 and suggest that they could be used for immunization of individuals with compromised CD4+ T-cell function.

AIDS Vaccines↗
PubMed1994

Exploratory analysis of multiple sequence alignments using phylogenies.

The significance of an alignment between two sequences can be determined using well-known techniques but cannot be easily evaluated with multiple alignments due to the computational complexity. Therefore multiple alignment algorithms may produce an alignment between sequences even when they have little homology with other sequences. A program is presented that makes use of a phylogeny to explore the implications of an alignment. Using the phylogeny, branch lengths are inferred and a search is conducted for regions of unusually rapid or slow rates of change given the observed rates in the rest of the sequence. A very rapid rate of change can be due to either poor homology or due to rapid divergence because of selection. Phylogenies are calculated using either the neighbor joining algorithm of Saitou and Nei (Mol. Biol. Evol., 4, 406-425, 1987) or a phylogeny supplied by the user. The program also permits randomization of subsections of the sequences to determine the significance of the multiple alignment for these individual regions. The combination of these two simple methods permits rapid and interactive exploration of multiple sequence alignments.

Algorithms↗
PubMed1994

The potential for recruiting immune responses toward type 1 or type 2 T cell help.

The design of vaccine strategies in general, and those for malaria in particular, need to take into account the balance of T helper subsets (TH) they induce. The TH1 cells, which secrete interferon-gamma and interleukin-2 (IL-2), are associated with cell-mediated immunity (CMI), rather than humoral responses, and afford protection against intracellular infections, including those caused by parasites. In contrast, the TH2 cells secrete IL-4, IL-5, and IL-10, elicit high titer antibody responses, provide poor CMI, and are often correlated with susceptibility to infection. Depending on the type of TH cell bias required, it is possible to manipulate the immune response to a protein/peptide by 1) using different adjuvants, 2) conjugating the protein to various carriers, 3) immunizing in the presence of cytokines, or 4) using alternative routes of administration. To apply these approaches to malarial vaccines, it is necessary to identify which stage(s) of the parasite to target and what type of TH cell bias is protective against that particular stage. We favor using carriers such as Brucella abortus, which focus the antigen on a specific particle and which can trigger a TH1 cell response.

Adjuvants, Immunologic↗
PubMed1993

Fusion of human B cell lines with HIV-1 envelope-expressing T cells is enhanced by antigen-specific Ig receptors. Possible mechanism for elimination of gp120-specific B cells in vivo.

The possible contribution of Ag-specific Ig receptors on B cells to syncytium formation with HIV-1 envelope (env)-expressing cells was examined. A unique model system was designed that used anti-TNP/TNP interactions between a panel of TNP-specific human B cell lines and TNP-haptenated HIV-1 env-expressing T cells. The prototype B cell line 1:13 (CD4dull) produced few syncytia with vaccinia gp120/41-infected CD4- T cell effectors. However, TNP-haptenation of the HIV-1 env-expressing cells resulted in a five- to 10-fold increase in syncytium formation. The "enhanced" syncytia were blocked by OKT4A mAb, soluble CD4, anti-TNP serum, and TNP-BSA, suggesting a role for both CD4 and Ig receptors. In contrast, the number of syncytia formed between CD4+ CEM T cells and TNP-haptenated effectors was reduced by 30 to 40%, compared with the unhaptenated effectors, suggesting that a fraction of the TNP haptens bound close to the CD4 binding regions on the gp120 envelope, which was confirmed by other experiments. The possibility that B cells specific for the CD4 binding site on HIV-1 gp120 may be involved in syncytium formation with HIV-1 env-expressing cells was tested by screening a panel of five hybrid B cell lines from HIV-1-seropositive individuals. One of these lines produced anti-gp120 antibodies, which bound near the CD4 binding site, and also formed syncytia with HIV-1 env-expressing cells. This study suggests that, in addition to CD4 receptors, certain B cell Ig receptors that bind to gp120 may induce conformational changes leading to cell fusion and their elimination.

B-Lymphocytes↗
PubMed1993

Lipopolysaccharide from Brucella abortus behaves as a T-cell-independent type 1 carrier in murine antigen-specific antibody responses.

In order to determine the carrier nature of lipopolysaccharide from Brucella abortus (LPS-BA) in evoking humoral responses, normal and immunodeficient mice were immunized with trinitrophenyl (TNP)-conjugated LPS-BA (TNP-LPS-BA) and the responses were compared with those to known T-dependent and T-independent antigens. TNP-LPS-BA, like T-independent type 1 (TI-1) antigens such as TNP-BA and TNP-LPS from Escherichia coli (TNP-LPS-EC), generated anti-TNP responses in BALB/c, athymic BALB/c nu/nu, and CBA/N mice. In contrast, N-2,4-dinitrophenyl-beta-alanylglycylglycyl-substituted keyhole limpet hemocyanin, a typical T-dependent antigen, was not immunogenic in athymic mice, and TNP-Ficoll (T-independent type 2) was ineffective in eliciting humoral responses in CBA/N mice. These results indicate that LPS from B. abortus acts as a TI-1 carrier in generating antibody responses. In C3H/HeJ mice, TNP-LPS-BA generated higher-titer immunoglobulin G1 (IgG1), IgG2a, and IgG2b anti-TNP antibodies than TNP-LPS-EC. Compared with those from BALB/c mice, pure resting B cells isolated from C3H/HeJ mice exhibited a 30-fold lower proliferative response to LPS-EC, whereas the LPS-BA response was reduced to a lesser extent (5-fold). This suggests that the disparity observed in antibody titers was due to different abilities of LPS from B. abortus and E. coli to stimulate C3H/HeJ B cells. The ability of LPS from B. abortus to act as a carrier in generating humoral immune responses indicates that LPS-BA can be substituted for whole B. abortus organisms in vaccine development.

Animals↗