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V F de la Cruz

Publications and source records attributed to V F de la Cruz.

At least 19 recordsLinked to original sources
PubMed1991

New use of BCG for recombinant vaccines.

BCG, a live attenuated tubercle bacillus, is the most widely used vaccine in the world and is also a useful vaccine vehicle for delivering protective antigens of multiple pathogens. Extrachromosomal and integrative expression vectors carrying the regulatory sequences for major BCG heat-shock proteins have been developed to allow expression of foreign antigens in BCG. These recombinant BCG strains can elicit long-lasting humoral and cellular immune responses to foreign antigens in mice.

Animals↗
PubMed1991

Identifying antigenic T-cell sites.

Computer algorithms that have been used successfully on protein sequences for the prediction of antigenic T-cell sites have been collected into a single computer software package called TSites.

Algorithms↗
PubMed1991

Development of BCG as a live recombinant vector system: potential use as an HIV vaccine.

Bacille Calmette-Guèrin (BCG), a live attenuated tubercle bacillus, is currently the most widely used vaccine in the world. Because of its unique characteristics, including low toxicity, adjuvant potential, and long-lasting immunity, BCG represents a novel vaccine vehicle with which to deliver protective antigens of multiple pathogens. We have developed episomal and integrative expression vectors employing regulatory sequences of major BCG heat shock proteins for stable maintenance and expression of foreign antigens in BCG vaccine strains (22). Shuttle plasmids capable of autonomous replication in Escherichia coli and BCG were constructed with a DNA cassette containing a minimal replicon derived from the Mycobacterium fortuitum plasmid pAL5000. Efficient and stable chromosomal integration of recombinant plasmids into BCG was achieved using a DNA segment containing the mycobacteriophage L5 attachment site and integrase coding sequence. Using the BCG hsp60 and hsp70 stress gene promoters, we were able to express Escherchia coli beta-galactosidase to levels in excess of 10% of total cell protein. The major antigens of HIV-1 gag, pol, and env were also stably expressed using our vector systems. The recombinant BCG elicited long-lasting humoral and cellular immune responses to these antigens in mice. Antibody responses to beta-galactosidase using as few as 200 colony-forming units were detected 6 weeks after immunization, and titers (1:30,000) were sustained for more than 10 weeks. Cellular immune responses, of both cytotoxic T cell (CTL) and helper T lymphocytes, were detected to beta-galactosidase. CTL responses were also induced to the HIV-1 envelope protein. Thus, we have demonstrated stable recombinant antigen expression, processing, and presentation using our recombinant BCG vector system. This live recombinant vector system shows promise as a universally applicable and safe vaccine vehicle for protection against various infectious diseases.

AIDS Vaccines↗
PubMed1991

Immunization against parasites: bridging the gap between attenuated and non-living vaccines.

While great progress has been made in the last decade in defining parasite antigens which are targets of host protective responses, only limited success has been achieved in the use of these molecules as effective vaccines. A consistent problem is the failure of the candidate immunogens to induce levels of protection comparable to those obtained with attenuated vaccines against the same organisms. One explanation is that the purified or recombinant molecules employed have not been presented in a form or route which induces the correct T cell or cytokine response necessary for protection. As summarized in this overview, optimal immunization with attenuated vaccines is associated with characteristic patterns of T cell subset and cytokine induction and in at least several examples has been shown to be altered by exogenous cytokine. We hypothesize that cytokine manipulation may offer a useful strategy for improving the action of existing nonliving vaccines. The gap in efficacy between attenuated and dead vaccines could also be bridged by the use of live recombinant vaccine vectors. We have previously reported that paramyosin admixed with BCG can induce partial protection against Schistosoma mansoni in mice. Our preliminary results in the construction and testing of a recombinant BCG vector incorporating schistosome paramyosin are described.

Animals↗
PubMed1990

Mapping the fine specificity of a cytolytic T cell response to HIV-1 nef protein.

Epitope mapping of a MHC class I-restricted cytotoxic T cell response to nef, a regulatory protein of HIV, was performed with fresh PBMC from HIV-seropositive donors and target cells pulsed with a panel of overlapping peptides of the nef protein. These nef-specific CTL recognized a synthetic peptide of 10 residues derived from a nonamphipathic, highly conserved region of the nef protein in association with the HLA A3.1 molecule. Using human cell transfectants expressing mutations of the A3 molecule, we demonstrated that the amino acid at position 152 of the A3.1 molecule appears to be critical for detection of this response. Thus, rapid analysis of the epitopes of HIV proteins stimulating CTL responses can be achieved using a combination of fresh donor PBMC and target cells pulsed with synthesized peptides.

Amino Acid Sequence↗
PubMed1988

Lack of cross-reactivity between variant T cell determinants from malaria circumsporozoite protein.

The discovery of polymorphism in the T cell determinants of the protein that covers the surface of malaria sporozoites, the circumsporozoite protein (CSP), may have a negative effect on the course of development of a sporozoite-derived anti-malaria vaccine. Comparison of CSP gene sequences from Plasmodium falciparum suggests, based on the lack of silent (i.e., synonymous) substitutions, that polymorphism is being biologically selected for in the field. Thus, variation in T cell determinant sequences may actually be a means of immune evasion. The central question addressed here is whether or not the natural polymorphisms found in three identified T cell determinants in the CSP gene of P. falciparum are immunologically significant with regard to T cell stimulation. In support of the immune evasion hypothesis, we show here that animals immunized with peptides based on one sequence (i.e., the 7G8 isolate) will not significantly respond when challenged with variant peptides based on other CSP sequences (i.e., the LE5 and We1 isolates). Polymorphism in T cell determinants thus indicates that infection with sporozoites will not necessarily boost immune (antibody help and/or proliferative) responses stimulated by prior infections or by a particular vaccine construct based on these determinants. The implications of these findings in regard to vaccine development are discussed.

Amino Acid Sequence↗
PubMed1988

Circumsporozoite protein gene from Plasmodium brasilianum. Animal reservoirs for human malaria parasites?

We describe here the sequence of the circumsporozoite protein gene of the monkey malaria parasite Plasmodium brasilianum and show that the immunodominant repeat domain is the same as that of the human malaria parasite, Plasmodium malariae. The immunodominant epitope on the surface of sporozoites of a third species of human malaria parasite has, therefore, been identified. This genetic based data and the biological similarities between P. brasilianum and P. malariae support their putative zoonotic/anthroponotic relationship. We also show that an ape malaria parasite, Plasmodium reichenowi, and the human malaria parasite, Plasmodium falciparum, have a similar relationship. The implications of these observations are discussed with respect to vaccine development.

Amino Acid Sequence↗
PubMed1988

Immunogenicity and epitope mapping of foreign sequences via genetically engineered filamentous phage.

Repeat regions of the circumsporozoite protein gene of Plasmodium falciparum were cloned into the pIII gene of a filamentous phage. These genetically engineered filamentous phage display the recombinant proteins on their surface. We demonstrate that they are both antigenic and immunogenic in rabbits. The recombinant phage were shown to be useful as a source of antigen for this scarce malaria protein, for producing carrier-hapten conjugates for obtaining immunological reagents in rabbits, and for B epitope mapping. In addition, in mice the antibody response to the cloned antigens seems to be controlled by immune response genes. Therefore this system also has the potential for use in helper T cell epitope mapping using inbred mouse strains. This advantage will be of use in vaccine development.

Animals↗
PubMed1988

Parasite polymorphism present within minimal T cell epitopes of Plasmodium falciparum circumsporozoite protein.

Identification of T cell antigenic sites is critical for antisporozoite malarial vaccine design. Here, we present data that define two minimal functional T cell sites present in an immunogenic domain of the circumsporozoite protein of Plasmodium falciparum. These two sites overlap one another and correlate with polymorphic regions of the molecule. This suggests that these polymorphisms may be a result of pressure from immune T cells.

Amino Acid Sequence↗
PubMed1988

Structure of the circumsporozoite gene of Plasmodium malariae.

The sequence of the gene encoding the circumsporozoite protein of Plasmodium malariae was determined. The central immunodominant region of the protein consists of 45 copies of the sequence Asn-Ala-Ala-Gly and 6 copies of the sequence Asn-Asp-Ala-Gly. The CSP of the monkey parasite Plasmodium brasilianum contains the same repetitive sequences. Further comparison of the two genes in regions outside the immunodominant domains reveals only three nucleotide differences and each results in an amino acid change. One is centered in a putative T-cell determinant bearing region, the second is in the putative liver binding site, and the third is part of a degenerate repeat at the start of the immunodominant region.

Amino Acid Sequence↗
PubMed1988

Variation among circumsporozoite protein genes from rodent malarias.

We investigated the effect of long term passage of parasites in naive animals on the circumsporozoite protein (CSP) gene of Plasmodium yoelii. The CSP gene sequence was determined from a non-lethal cloned line of P. yoelii and compared to the CSP gene sequence from a lethal strain of P. yoelii. The two parasite lines were originally derived from the same isolate, but were separated 17 years ago followed by continued passage. The sequence of the CSP gene and its surroundings from the non-lethal line remains identical to that from the lethal isolate except for a deletion within the repeated central domain. This result contrasts with the results obtained by sequencing a number of clones from different geographical field isolates of Plasmodium falciparum where there appears to be rapid accumulation of sense mutations within putative functional domains. These observations are consistent with the suggestion that strong biological pressure in a field environment results in selection of parasite types on the basis of different CSP gene sequences.

Amino Acid Sequence↗
PubMed1988

Primary sequences of two small subunit ribosomal RNA genes from Plasmodium falciparum.

We have determined the complete sequence of two structurally distinct 18S ribosomal RNA genes from the malarial parasite Plasmodium falciparum. S1 nuclease analyses demonstrate that only one of the genes is represented in stable rRNA populations isolated from blood-stage parasites. Comparisons of homologous rRNA genes from Plasmodium berghei and P. falciparum reveal that they are identical at 86% of their positions. From comparisons of the Plasmodium genes to that of humans, it was possible to design genus-specific as well as species-specific oligonucleotide probes that can be used to distinguish the parasite 18S ribosomal RNA from that of its host. The utilization of these probes as diagnostic reagents is discussed.

Animals↗
PubMed1988

Evolution of parasitism: kinetoplastid protozoan history reconstructed from mitochondrial rRNA gene sequences.

A phylogenetic tree for the evolution of five representative species from four genera of kinetoplastid protozoa was constructed from comparison of the mitochondrial 9S and 12S rRNA gene sequences and application of both parsimony and evolutionary parsimony algorithms. In the rooted version of the tree, the monogenetic species Crithidia fasciculata is the most deeply rooted, followed by another monogenetic species, Leptomonas sp. The three digenetic species Trypanosoma cruzi, Trypanosoma brucei, and Leishmania tarentolae branch from the Leptomonas line. The substitution rates for the T. brucei and T. cruzi sequences were 3-4 times greater than that of the L. tarentolae sequences. This phylogenetic tree is consistent with our cladistic analysis of the biological evidence including life cycles for these five species. A tentative time scale can be assigned to the nodes of this tree by assuming that the common ancestor of the digenetic parasites predated the separation of South America and Africa and postdated the first fossil appearance of its host (inferred by parsimony analysis). This time scale predicts that the deepest node occurred at 264 +/- 51 million years ago, at a time commensurate with the fossil origins of the Hemiptera insect host. This implies that the ancestral kinetoplastid and its insect host appeared at approximately the same time. The molecular data suggest that these eukaryotic parasites have an evolutionary history that extends back to the origin of their insect host.

Animals↗