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Publications and source records attributed to C Sia.
A library of conformationally constrained reverse turn mimetics of the HIV V3 loop was screened against the mAb 50.1. Addition of a Th-cell epitope to the reverse turn template provided an immunogen which, when immunized (either in alum or IFA), generated a high titer, highly specific response to HIV gp 120.
An important goal of structural biochemistry is the reduction of complex molecules to small functional units that are amenable to high-resolution structural analysis and rapid modification. The dissection of multidomain proteins into small synthetic conformationally restricted components is an important step in the design of low molecular weight nonpeptides that mimic the activity of the native protein. Mimetics of critical functional domains might possess beneficial properties in comparison to the intact proteinaceous species with regard to specificity and therapeutic potential, and are valuable probes for the study of molecular recognition events, and the development of novel vaccines and pharmaceuticals.
Monocytes/macrophages and neutrophils can respond to endotoxin via a high-affinity receptor (CD14), requiring low levels of LPS (< 1 ng/ml) as well as through another pathway(s) that requires high levels of LPS (> 10 ng/ml). Both pathways result in the secretion of high levels of cytokines, such as TNF-alpha, and the up-regulation of various other effector molecules. To further define the activation of cells by LPS via a pathway that does not involve CD14, we have used an experimental model that distinguishes CD14-dependent from CD14-independent responses using saturating amounts of an anti-CD14 Ab to block the CD14-dependent response. Analysis of the ability of various individuals to respond to LPS via via both the CD14-dependent and CD14-independent pathways shows that adults can respond via both pathways; furthermore, in the presence of 100 ng of LPS/ml, the primary response is CD14 independent. In contrast to the response by adults, neonates can only respond via the CD14-dependent pathway. Further analysis has shown that the CD14-independent pathway requires a non-CD14 plasma protein present in adult plasma that is either missing or nonfunctional in neonate (cord) plasma.
Genetically engineered, noninfectious HIV-1-like particles containing processed envelope glycoproteins represent potential candidate immunogens for a vaccine against HIV-1. However, since the gp120 glycoprotein is known to be rapidly lost from the surface of infected cells and purified virions as a result of its low-affinity interaction with gp41, shedding of this extracellular subunit could compromise the immunogenic potential of particle-based HIV-1 vaccine candidates. In this study, we demonstrate for the first time the feasibility of producing fully assembled HIV-1-like particles containing only unprocessed gp160 glycoproteins. Monkey kidney Vero cells were transfected with an inducible, human metallothionein-based expression vector containing most of the HIV-1LAI coding sequences that were genetically modified to introduce safety mutations and destroy the major cleavage site of the HIV-1 envelope glycoprotein. A stably-transfected cell line was isolated and shown to secrete HIV-1-like particles containing unprocessed gp160. Immunization with these particles induced HIV-1 cross-neutralizing, syncytium-inhibiting and env-CD4 blocking antibodies. Thus, these novel HIV-1-like particles represent alternative candidate immunogens for the development of a particle-based AIDS vaccine.
To identify the B- and T-cell epitopes of P1 of Haemophilus influenzae type b, 13 peptides covering 90% of the protein were chemically synthesized. Mouse, guinea pig, and rabbit antisera raised against purified native P1 were tested for their reactivities against the peptides in peptide-specific enzyme-linked immunosorbent assays (ELISAs). Six immunodominant linear B-cell epitopes were mapped to residues 103 to 137, 189 to 218, 248 to 283, 307 to 331, 384 to 412, and 400 to 437 of the mature P1 protein. When P1 peptides were screened for their reactivities with three human convalescent-phase serum specimens, peptides corresponding to residues 39 to 64, 226 to 253, and 400 to 437 reacted strongly with the antisera. Four regions (residues 39 to 64, 226 to 253, 339 to 370, and 400 to 437) contained murine T-cell epitopes. Rabbit antipeptide antisera were tested for their reactivities with the immunizing peptides and P1 protein by ELISA and immunoblots. All anti-P1 peptide antisera except those raised against peptide HIBP1-8 (residues 279 to 312) or HIBP1-8-keyhole limpet hemocyanin conjugate were shown to be specific for their respective immunizing peptides by ELISA. In addition, rabbit antisera raised against the synthetic peptides corresponding to residues 1 to 29, 39 to 64, 103 to 137, 189 to 218, 226 to 253, 248 to 283, 307 to 331, and 400 to 437 of the mature P1 protein recognized the P1 protein from both typeable and nontypeable isolates. These results suggest that these peptides contain epitopes highly conserved among typeable and nontypeable strains of H. influenzae. However, none of the antipeptide antisera have bactericidal activity, nor were they protective against H. influenzae type b in the infant rat model of bacteremia.
A 134-mer peptide corresponding to the N-terminal sequence of p24 (residues 146-279 of the gag gene product of the LAV strain) was chemically synthesized using highly optimised protocols on an ABI 430A synthesizer. The crude peptide was obtained by treating the peptide-resin with HF, then purified by a combination of size exclusion and RP-HPLC. One hundred milligram of 90% pure 134-mer can be obtained within a month. Both mice and rabbit polyclonal antisera raised against a commercial preparation of recombinant p24, and a pooled sera from HIV-1 infected individuals reacted strongly with the 134-mer peptide in ELISA. Both mice and rabbits immunized with the free peptide emulsified in Freund's complete adjuvant generated strong anti-peptide and anti-p24 antibody responses as judged by immunoblots and ELISAs. Immunodominant epitopes were mapped to residues 201-227 (LKETINEEAAEWDRVHPVHAGPIAPG). These B-cell epitopes had previously been identified by mouse monoclonal antibodies raised against HIV-1 virus or gag gene products. Furthermore, murine T-cell lines generated against the 134-mer peptide were found to respond to two short peptides, P24B (residues 195-215) and P24D1 (residues 268-279). These two T-cell epitopes were previously reported as human helper T-cell and CTL epitopes, respectively. These results clearly indicate that the synthetic 134-mer peptide could elicit both T- and B-cell responses to HIV-1 similar to those obtained with the natural viral gag protein, and could be useful for the development of a synthetic HIV vaccine.
Haemophilus influenzae type b is a major cause of bacterial meningitis in young children. Antibodies against the outer membrane protein P2 are protective in the infant rat model of bacteremia. To identify conserved, surface-exposed, and protective epitopes of P2, 17 overlapping peptides covering the entire sequence of the protein were synthesized. Antisera from mice, guinea pigs, and rabbits raised against chromatographically purified P2 were tested for their reactivities to the peptides by enzyme-linked immunosorbent assays (ELISA). Three major linear immunodominant B-cell epitopes were mapped to residues 53 to 81, 241 to 265, and 314 to 341 of mature P2. Human convalescent-phase antisera also reacted strongly with these three epitopes. Rabbit antisera against all peptide-keyhole limpet hemocyanin conjugates except two peptides containing residues 8 to 19 and 302 to 319 recognized the corresponding peptides in ELISA and reacted with P2 on immunoblots. Immunization with all unconjugated peptides, except the 19 N-terminal residues, induced very strong peptide-specific antibody responses, and these antisera reacted with P2 on immunoblots. Rabbit antisera raised against peptides corresponding to residues 1 to 14, 125 to 150, 193 to 219, and 241 to 319 also recognized P2 purified from H. influenzae nontypeable isolates. Identification of these immunodominant B-cell epitopes and conserved regions is a first step toward the rational design of a universal H. influenzae vaccine.
To design an optimized synthetic vaccine against whooping cough, we have studied the biological and immunological properties of three peptides of the S2 subunit and nine overlapping synthetic peptides covering the entire sequence of the S3 subunit of pertussis toxin (PT). Synthetic peptides corresponding to sequences 18 to 41, 78 to 108, 134 to 154, and 149 to 176 of S3 were found to be consistently capable of stimulating the proliferation of PT-specific T-cell lines primed with pertussis toxoid in both BALB/c and A/J strains of mice. All synthetic peptides were recognized by rabbit antisera raised against PT or pertussis toxoid. Both S2 and S3 peptide-keyhole limpet hemocyanin (KLH) conjugates in the presence of complete Freund's adjuvant induced peptide-specific antibody responses in rabbits, and the antisera raised against S2(1-23), S3(18-41), S3(37-64), and S3(149-176) peptide-KLH conjugates cross-reacted with both subunits in the immunoblots. All antisera except those against S2(123-154) and S3(103-127) reacted with native PT in an enzyme-linked immunosorbent assay (ELISA) with PT directly coated onto microtiter wells. In contrast, antisera raised against S2(123-154), S3(1-23), S3(18-41), S3(37-64), S3(60-87), and S3(103-127) peptide-KLH conjugates recognized native PT in a fetuin-PT capture ELISA. S2(78-98), S3(1-23), and S3(149-176) peptide-KLH conjugates elicited good PT-neutralizing antibody responses as judged by the antitoxin CHO cell assay. Identification of these B-cell neutralization epitopes and T-cell immunodominant determinants represents a first step towards the rational design of a synthetic vaccine against whooping cough.
Noninfectious human immunodeficiency virus type 1 (HIV-1) viruslike particles containing chimeric envelope glycoproteins were expressed in mammalian cells by using inducible promoters. We engineered four expression vectors in which a synthetic oligomer encoding gp120 residues 306 to 328 (amino acids YNKRKRIHIGP GRAFYTTKNIIG) from the V3 loop of the MN viral isolate was inserted at various positions within the endogenous HIV-1LAI env gene. Expression studies revealed that insertion of the heterologous V3(MN) loop segment at two different locations within the conserved region 2 (C2) of gp120, either 173 or 242 residues away from the N terminus of the mature subunit, resulted in the secretion of fully assembled HIV-like particles containing chimeric LAI/MN envelope glycoproteins. Both V3 loop epitopes were recognized by loop-specific neutralizing antibodies. However, insertion of the V3(MN) loop segment into other regions of gp120 led to the production of envelope-deficient viruslike particles. Immunization with HIV-like particles containing chimeric envelope proteins induced specific antibody responses against both the autologous and heterologous V3 loop epitopes, including cross-neutralizing antibodies against the HIV-1LAI and HIV-1MN isolates. This study, therefore, demonstrates the feasibility of genetically engineering optimized HIV-like particles capable of eliciting cross-neutralizing antibodies.
A proviral fragment from human immunodeficiency virus type 1 (HIV-1) (LAV-1BRU) containing only protein-coding information, was expressed in COS cells using constitutive promoters in transient and stable transfection experiments. The presence of viruslike particles in cell supernatants was verified by Western blot analysis, density gradient centrifugation, and electron microscopy. Transfection of Vero cells with a similar construct employing the human metallothionein promoter led to the isolation of stable cell lines exhibiting inducible viruslike particle expression in response to cadmium chloride treatment. Induction ratios for viruslike particle expression were in excess of 1000-fold with production levels of p24 core antigen as high as 0.6 mg/L per 24 h. HIV-1 viruslike particles were immunogenic in mice, leading to strong envelope and core-specific humoral responses after two immunizations. The development of stable cell lines expressing significant quantities of HIV-1 viruslike particles offers an alternative to the use of live virus vectors for the production and evaluation of particle-based AIDS vaccines.
Recent studies indicate that the gag gene products may play a crucial role in the immune response against HIV infection since clinical progression to AIDS is associated with a reduction in the level of circulating antibodies to gag p24 and antibodies raised against p17 peptide can inhibit HIV1 infection in vitro. Using conventional structure prediction algorithms for T-cell and B-cell epitopes, we have selected and chemically synthesized several gag peptides. In particular, an unconjugated HIV1-p24 peptide containing both B- and T-cell epitopes in tandem plus Freund's adjuvant induced a strong antibody response in both mice and rabbits against p24 and its precursor p55 as judged by immunoblotting. In addition, the peptide presented in the appropriate MHC context was shown to be highly stimulatory for p24 specific murine T-cell clones.
Methodology was established to analyze T lymphocytes and T cell subsets with monoclonal antibodies on microsamples of blood obtained by heel puncture in infants. Results in 39 high-risk infants indicated that during the early neonatal period they had a higher percentage of T4-bearing cells and a lower percentage of T8 antigen-positive cells as compared with adults. These values progressively approached adult values, and at 3 to 7 months the T cell subsets and the T4/T8 ratios were within the adult range. A significant inverse relationship was noted between the number of blood transfusions given to the infants between 2 and 12 weeks of age and the corresponding T4/T8 ratio. These findings suggest that interpretation of T cell subsets in frequently transfused infants should take into consideration not only the age of the infant but also the possible influence of transfusions per se on the distribution of T cell subsets in the peripheral blood.
Human T lymphocytes bear characteristic membrane antigens which allow for identification on these cells and their subpopulations with monoclonal antibody reagents directed against the specific cell-surface antigens. During a study of T lymphocyte subpopulations in a group of 41 infants from a high risk nursery, three of the seven black infants studied were found to be missing cells reactive with the monoclonal antibody OKT4 which identifies the helper-inducer subset of T cells. Immunological evaluation of these infants and study of their family members revealed that the OKT4 non-reactive lymphocytes reacted normally with another antibody, OKT4A, which also identifies the helper-inducer subset of T cells. The deficiency of the antigen recognized by the OKT4 antibody appeared not to reflect T cell immaturity and was not associated with obvious immunodeficiency. The OKT4 negative phenotype appeared to be transmitted in an autosomal recessive mode. Our studies suggest that heterozygosity for this phenotype is relatively common among the black population and that heterozygotes are not easily distinguishable from the random population on the basis of lymphocyte reactivity with the OKT4 monoclonal antibody.
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Thirty percent of 37 consecutive surviving premature infants weighing 1,000 gm or less at birth were noted to develop inguinal hernias. Incarceration occurred in two infants. One infant suffered a cardiac arrest during repair of the hernia. In view of the increased survival now being reported in these tiny prematures, it is important that physicians recognize the high incidence and potential hazards of this congenital anomaly in surviving premature infants weighing 1,000 gm or less at birth.