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1.
Cytotechnology ; 35(2): 115-25, 2001 Mar.
Article in English | MEDLINE | ID: mdl-19003288

ABSTRACT

The density of viable cells in a culture results from a balance between cell proliferation and cell death. The aim of this study was to characterize and compare these two phenomena in Vero cell cultures in one serum containing medium (ScA) and one serum free medium (SfB) in bioreactors. Cell growth was evaluated by cell counting(after crystal violet staining) and cell cycle analysis. Necrosis and apoptosis were characterized and quantified by measuring the release of LDH, trypan blue exclusion,annex in V-FITC/PI staining and TUNEL assay. ScA supported a higher maximal viable-cell density(2.3 x 10(6) vs. 1.8 x 10(6) cells ml(-1)). However, cell cycle analysis showed that cell division was more active in SfB than in ScA. LDH release in the supernatant increased much earlier in SfB than in ScA (one vs. five days), but trypan blue counts showed no apparent difference in the viability of the cultures. Apoptosis, evidenced by annexin V-FITC/PI staining, could be detected in the population of suspension cells detached from microcarriers, but not among adherent cells; positivity of the TUNEL assay occurred later than that of the annexin V-FITC/PI staining. Our data indicate that the lower cell yield in SfB,compared with that in ScA, results from a higher cell death rate. Apparently, cells die from apoptosis followed by secondary necrosis.

3.
AIDS Res Hum Retroviruses ; 15(7): 633-45, 1999 May 01.
Article in English | MEDLINE | ID: mdl-10331442

ABSTRACT

A live recombinant canarypox vector expressing HIV-1 gpl20 MN tm/gag/protease LAI (ALVAC-HIV, vCP205) alone or boosted by a p24E-V3 MN synthetic peptide (CLTB-36) was tested in healthy volunteers at low risk for HIV infection for their safety and immunogenicity. Both antigens were well tolerated. ALVAC-HIV (vCP205) induced low levels of neutralizing antibodies against HIV-1 MN in 33% of the volunteers. None of them had detectable neutralizing antibodies against a nonsyncytium-inducing HIV-1 clade B primary isolate (Bx08). After the fourth injection of vCP205, CTL activity was detected in 33% of the volunteers and was directed against Env, Gag, and Pol. This activity was mediated by both CD4+ and CD8+ lymphocytes. On the other hand, the CLTB-36 peptide was poorly immunogenic and induced no neutralizing antibodies or CTLs. Although the ALVAC-HIV (vCP205) and CLTB-36 prime-boost regimen was not optimal, further studies with ALVAC-HIV (vCP205) are warranted because of its clear induction of a cellular immune response and utility as a priming agent for other subunit antigens such as envelope glycoproteins, pseudoparticles, or new peptides.


Subject(s)
AIDS Vaccines/adverse effects , AIDS Vaccines/immunology , Avipoxvirus/immunology , HIV Antibodies/biosynthesis , HIV-1/immunology , Vaccines, Synthetic/adverse effects , Vaccines, Synthetic/immunology , Adult , Amino Acid Sequence , Avipoxvirus/genetics , Female , Genetic Vectors , HIV Antibodies/blood , HIV Core Protein p24/chemistry , HIV Core Protein p24/immunology , HIV Envelope Protein gp120/adverse effects , HIV Envelope Protein gp120/chemistry , HIV Envelope Protein gp120/immunology , Humans , Immunization Schedule , Immunization, Secondary , Lymphocyte Activation , Male , Middle Aged , Molecular Sequence Data , Peptides/chemical synthesis , Peptides/chemistry , Peptides/immunology , T-Lymphocytes/immunology , T-Lymphocytes, Cytotoxic/immunology
5.
AIDS Res Hum Retroviruses ; 14(12): 1023-34, 1998 Aug 10.
Article in English | MEDLINE | ID: mdl-9718117

ABSTRACT

The fine specificity of the anti-V3 antibody responses induced in chimpanzees immunized by various human immunodeficiency type 1 (HIV-1) candidate vaccines and challenged by heterologous strains of HIV-1 was analyzed by enzyme-linked immunosorbent assay (ELISA) and Pepscan epitope mapping. Two chimpanzees immunized with the recombinant canarypox virus ALVAC-HIV (vCP125) expressing gp160MN and boosted with purified gp160MN/LAI alone, then with both immunogens in combination, were not protected against challenge with HIV-1 SF2. Their sera mainly recognized one epitope of the V3 loop, located in the NH2-terminal half. By contrast, immunization of two other chimpanzees with purified gp160MN/LAI and boosting with a synthetic V3MN peptide elicited a strong anti-V3 antibody response with a broader specificity directed against multiple epitopes all along the V3 loop. These chimpanzees were protected against infection by HIV-1 SF2. However, when these two chimpanzees were challenged later with a HIV-1 clade E strain virus, they became infected. We failed to detect any reactivity with the peptide of the ectodomain of gp41 of sera harvested after immunization with the various immunogens or after challenge with HIV-1 SF2 or HIV-1 90CR402. These results demonstrated that anti-V3 antibodies with a restricted fine specificity were induced in chimpanzees immunized with gp160 purified or expressed by recombinant canarypox confirming our previous results obtained in three different species (human, guinea pig and, macaque). In contrast, a boost with the V3 peptide broadened antibody responses, suggesting that the mode of presentation of the V3 loop to the immune system strongly influences the epitope specificity of the resulting antibody response.


Subject(s)
AIDS Vaccines/immunology , Antibody Specificity , HIV Antibodies/biosynthesis , HIV Envelope Protein gp120/immunology , Peptide Fragments/immunology , Amino Acid Sequence , Animals , Enzyme-Linked Immunosorbent Assay , Epitopes/chemistry , Epitopes/immunology , HIV Antibodies/immunology , HIV-1/immunology , Male , Molecular Sequence Data , Pan troglodytes
6.
AIDS Res Hum Retroviruses ; 13(17): 1471-85, 1997 Nov 20.
Article in English | MEDLINE | ID: mdl-9390746

ABSTRACT

We analyzed the fine specificity of anti-V3 antibodies elicited in three different species (human, guinea pig, and macaque) by various HIV candidate vaccines. Following immunization with recombinant canarypox virus expressing gp160MN or with recombinant gp160MN/LAI, this antibody response was shown to be directed against the NH2-terminal region of the V3 loop. Although this response was increased by a prime-boost regimen using immunization with canarypox expressing gp160 followed by an rgp160 boost, its specificity remained restricted mainly to the recognition of this region of the V3 loop. Pepscan analysis of sera confirmed the results obtained by ELISA and allowed the definition of an immunodominant common binding site for these sera located within the sequence NKRKRIHIGPGR. In contrast to these results, a boost with the V3 peptide was shown to broaden the antibody response and pepscan analysis showed that sera from individuals boosted with the V3 synthetic peptide recognize determinants all along the V3 loop. Similar fine specificity of anti-V3 antibodies was obtained in human, guinea pig, and macaque following immunization by a prime-boost regimen using canarypox recombinants expressing gp160 or gp120 and purified rgp160. In contrast, a V3 synthetic peptide boost stimulated the production of antibodies that recognize multiple epitopes within the V3 loop. Because the induction of antibodies that recognize multiple sites in the V3 loop could be of major importance to neutralize different HIV isolates, these results may have implications for the design and selection of HIV candidate vaccines.


Subject(s)
AIDS Vaccines/immunology , Antibodies, Viral/immunology , Antibody Specificity , HIV Envelope Protein gp120/immunology , Peptide Fragments/immunology , Amino Acid Sequence , Animals , Enzyme-Linked Immunosorbent Assay , Epitope Mapping , Guinea Pigs , HIV Envelope Protein gp120/chemistry , Humans , Macaca , Molecular Sequence Data , Peptide Fragments/chemistry
7.
Virology ; 232(1): 98-104, 1997 May 26.
Article in English | MEDLINE | ID: mdl-9185593

ABSTRACT

To evaluate the potential protective efficacy of a live recombinant human immunodeficiency virus type 1 (HIV-1) canarypox vaccine candidate, two chimpanzees were immunized five times with ALVAC-HIV-1 vCP250, a recombinant canarypox virus that expresses the HIV-1[IIIB(LAI)] gp120/TM, gag, and protease gene products. One month after the last booster inoculation, the animals were challenged by intravenous injection of cell-associated virus in the form of peripheral blood mononuclear cells from an HIV-1[IIIB(LAI)]-infected chimpanzee. One chimpanzee with a neutralizing antibody titer to HIV-1[IIIB(LAI)] of 128 at the time of challenge was protected, whereas both the second animal, with a neutralizing antibody titer of 32, and a naive control animal became infected. At 5 months after challenge, the protected chimpanzee and a third animal, previously immunized with various HIV-1[MN] antigens, were given a booster inoculation. The two animals were challenged intravenously 5 weeks later with twenty 50% tissue culture infectious doses of cell-free HIV-1[DH12], a heterologous subtype B isolate. Neither chimpanzee had neutralizing antibodies to HIV-1[DH12], and neither one was protected from infection with this isolate. The immune responses elicited by vaccination against HIV-1[IIIB(LAI)] or HIV-1[MN] did not, therefore, protect the animals from challenge with the heterologous cell-free HIV-1[DH12].


Subject(s)
AIDS Vaccines/immunology , HIV Infections/prevention & control , Vaccines, Synthetic/immunology , Viral Vaccines/immunology , AIDS Vaccines/administration & dosage , Animals , Avipoxvirus/genetics , HIV Antibodies/biosynthesis , HIV Infections/immunology , HIV-1/immunology , Immunoenzyme Techniques , Male , Neutralization Tests , Pan troglodytes , Vaccines, Synthetic/administration & dosage , Viral Vaccines/administration & dosage
8.
J Virol ; 70(11): 8229-33, 1996 Nov.
Article in English | MEDLINE | ID: mdl-8892959

ABSTRACT

Generation of an effective vaccine against human immunodeficiency virus type 1 (HIV-1) must overcome problems associated with extensive genetic diversity. Although we previously reported vaccine-induced protection of chimpanzees against infection with an HIV-1 strain different from the one used to make the immunogens, both the HIV-1 vaccine and challenge strains were classified within subtype B. To determine whether the HIV-1-specific immunity elicited might also prevent infection by a strain of HIV-1 from a different clade, the same chimpanzees were given booster inoculations with the rgp160-MN/LAI (recombinant hybrid gp160 molecule) and V3-MN immunogens and then were challenged by intravenous inoculation of a comparable dose of a subtype E HIV-1 from the Central African Republic. Both animals became infected with the subtype E virus, indicating that intraclade vaccine-mediated protection does not predict interclade protection, at least in the context of intravenous challenge and the HIV-1 strains used. This study has important implications for planned phase III efficacy trials of similar vaccine preparations in Thailand where HIV-1 subtype B and E strains cocirculate.


Subject(s)
AIDS Vaccines/immunology , HIV Infections/prevention & control , HIV-1/immunology , Vaccines, Synthetic/immunology , Amino Acid Sequence , Animals , Disease Models, Animal , HIV Antibodies/blood , HIV Envelope Protein gp120/genetics , HIV-1/genetics , HIV-1/isolation & purification , Humans , Molecular Sequence Data , Pan troglodytes , Peptide Fragments/genetics , Sequence Homology, Amino Acid
9.
J Virol ; 70(8): 5519-24, 1996 Aug.
Article in English | MEDLINE | ID: mdl-8764064

ABSTRACT

The preparation of live, attenuated human influenza virus vaccines and of large quantities of inactivated vaccines after the emergence or reemergence of a pandemic influenza virus will require an alternative host cell system, because embryonated chicken eggs will likely be insufficient and suboptimal. Preliminary studies indicated that an African green monkey kidney cell line (Vero) is a suitable system for the primary isolation and cultivation of influenza A viruses (E. A. Govorkova, N. V. Kaverin, L. V. Gubareva, B. Meignier, and R. G. Webster, J. Infect. Dis. 172:250-253, 1995). We now demonstrate for the first time that Vero cells are suitable for isolation and productive replication of influenza B viruses and determine the biological and genetic properties of both influenza A and B viruses in Vero cells; additionally, we characterize the receptors on Vero cells compared with those on Madin-Darby canine kidney (MDCK) cells. Sequence analysis indicated that the hemagglutinin of Vero cell-derived influenza B viruses was identical to that of MDCK-grown counterparts but differed from that of egg-grown viruses at amino acid positions 196 to 198. Fluorescence-activated cell sorting analysis showed that although Vero cells possess predominantly alpha2,3 galactose-linked sialic acid, they are fully susceptible to infection with either human influenza A or B viruses. Moreover, all virus-specific polypeptides were synthesized in the same proportions in Vero cells as in MDCK cells. Electron microscopic and immunofluorescence studies confirmed that infected Vero cells undergo the same morphological changes as do other polarized epithelia] cells. Taken together, these results indicate that Vero cell lines could serve as an alternative host system for the cultivation of influenza A and B viruses, providing adequate quantities of either virus to meet the vaccine requirements imposed by an emerging pandemic.


Subject(s)
Influenza A virus , Influenza B virus , Influenza, Human/virology , Receptors, Virus/metabolism , Vero Cells/virology , Animals , Chlorocebus aethiops , Dogs , Flow Cytometry , Humans , Influenza A virus/metabolism , Influenza B virus/metabolism , Microscopy, Electron , Vero Cells/ultrastructure , Viral Vaccines , Virus Activation
11.
Vaccine ; 14(5): 428-34, 1996 Apr.
Article in English | MEDLINE | ID: mdl-8735555

ABSTRACT

Avian poxvirus recombinants undergo abortive replication in nonavian cells, yet can achieve expression of extrinsic gene products. Canarypox-vectored vaccines have been innocuous and immunogenic in several mammalian species. ALVAC-RG, a canarypox recombinant expressing the rabies glycoprotein gene, was inoculated intramuscularly into adult volunteers on days 0, 28, and 180. Sequential cohorts received 10(3.5), 10(4.5), and 10(5.5) 50% tissue culture infective doses (TCID50); additional volunteers received the standard human diploid cell rabies vaccine (HDCV) on the same schedule. Reactogenicity of ALVAC-RG was minimal. The lowest dose of ALVAC-RG induced little antibody to rabies virus by ELISA or rapid fluorescent focus inhibition test (RFFIT), but 10(4.5) and 10(5.5) TCID50 doses elicited significant responses in both assays. All recipients of 10(4.5) and 10(5.5) TCID50 of ALVAC-RG attained RFFIT values above the presumed protective level. Canarypox-specific immune responses did not inhibit boosting of rabies-specific antibodies by the day 180 dose of ALVAC-RG. T cell proliferation in response to inactivated rabies virus in vitro was similar in HDCV and ALVAC-RG recipients after the first and second doses, although HDCV yielded superior results after the third dose. ALVAC-RG was safe in humans, induced functional antibody to rabies glycoprotein, elicited cellular responses to rabies virus, and could be used successfully for booster dosing at a 6 month interval.


Subject(s)
Antigens, Viral , Avipoxvirus/genetics , Glycoproteins/immunology , Rabies Vaccines/immunology , Vaccines, Synthetic/immunology , Viral Envelope Proteins/immunology , Adolescent , Adult , Antibodies, Viral/blood , Female , Glycoproteins/adverse effects , Humans , Immunoglobulin G/blood , Lymphocyte Activation , Middle Aged , Rabies Vaccines/adverse effects , Vaccines, Synthetic/adverse effects , Viral Envelope Proteins/adverse effects
12.
AIDS Res Hum Retroviruses ; 11(12): 1479-86, 1995 Dec.
Article in English | MEDLINE | ID: mdl-8679292

ABSTRACT

The safety and the immunogenicity of a recombinant hybrid envelope glycoprotein MN/LAI (rgp160) followed by booster injections of a V3 (MN) linear peptide were evaluated in HIV-negative adults at low risk for HIV infection. Volunteers received either rgp160 in alum at 0, 1, and 6 months (group A), rgp160 at 0 and 1 months followed by V3 at 3 and 6 months formulated in alum (group B), or in Freund's incomplete adjuvant (FIA) (group C). Local and systemic reactions were mild to moderate and resolved within the first 72 hr after immunization. No significant biological changes (routine tests and autoantibodies) were observed. One month after the last injection in either group, neutralizing antibodies (NAs) against the HIV-1 MN isolate were detected in 4 of 5 (A), 7 of 10 (B), and 10 of 10 (C) subjects with significantly higher geometric mean titers in the latter group. Four of nine sera with the highest NA titers against MN weakly cross-neutralized the HIV-1 SF2 isolate; none had NA against the HIV-1 LAI strain or against a North American primary isolate. Specific lymphocyte T cell proliferation to rgp160 was detected in 92% of the subjects after the second injection of rgp160 and in 80% of them 12 months after the first injection. A weak and short-lived envelope-specific CD(4+)-mediated cytotoxic lymphocyte activity was detected at certain time points in few subjects. This prime-boost vaccine approach using rgp160 followed by a V3 peptide was safe in humans, and was able to elicit high levels of neutralizing antibodies and a strong and persistent T cell lymphoproliferative response to rgp160 and to V3. However, the neutralization response was restricted to the homologous HIV-1 strain and little env-specific cytotoxic activity was induced.


Subject(s)
AIDS Vaccines/immunology , Gene Products, env/genetics , Gene Products, env/immunology , HIV Envelope Protein gp120/immunology , Peptide Fragments/immunology , Protein Precursors/genetics , Protein Precursors/immunology , AIDS Vaccines/standards , Adult , CD4-Positive T-Lymphocytes/immunology , CD8-Positive T-Lymphocytes/immunology , Cytotoxicity, Immunologic , Female , HIV Antibodies/biosynthesis , HIV Envelope Protein gp160 , HIV-1/chemistry , HIV-1/genetics , Humans , Immunization Schedule , Lymphocyte Activation , Lymphocyte Count/methods , Male , Middle Aged , Neutralization Tests/methods , Random Allocation , Recombinant Proteins/immunology
13.
J Virol ; 69(10): 6239-48, 1995 Oct.
Article in English | MEDLINE | ID: mdl-7666524

ABSTRACT

The extraordinary genetic diversity of human immunodeficiency virus type 1 (HIV-1) is a major problem to overcome in the development of an effective vaccine. In the most reliable animal model of HIV-1 infection, chimpanzees were immunized with various combinations of HIV-1 antigens, which were derived primarily from the surface glycoprotein, gp160, of HIV-1 strains LAI and MN. The immunogens also included a live recombinant canarypox virus expressing a gp160-MN protein. In one experiment, two chimpanzees were immunized multiple times; one animal received antigens derived only from HIV-1LAI, and the second animal received antigens from both HIV-1LAI and HIV-1MN. In another experiment, four chimpanzees were immunized in parallel a total of five times over 18 months; two animals received purified gp160 and V3-MN peptides, whereas the other two animals received the recombinant canarypox virus and gp160. At 3 months after the final booster, all immunized and naive control chimpanzees were challenged by intravenous inoculation of HIV-1SF2; therefore, the study represented an intrasubtype B heterologous virus challenge. Virologic and serologic follow-up showed that the controls and the two chimpanzees immunized with the live recombinant canarypox virus became infected, whereas the other animals that were immunized with gp160 and V3-MN peptides were protected from infection. Evaluation of both cellular and humoral HIV-specific immune responses at the time of infectious HIV-1 challenge identified the following as possible correlates of protection: antibody titers to the V3 loop of MN and neutralizing antibody titers to HIV-1MN or HIV-1LAI, but not to HIV-1SF2. The results of this study indicate that vaccine-mediated protection against intravenous infection with heterologous HIV-1 strains of the same subtype is possible with some immunogens.


Subject(s)
AIDS Vaccines/immunology , Acquired Immunodeficiency Syndrome/immunology , DNA, Viral/analysis , HIV Antibodies/blood , HIV Antigens/immunology , HIV-1/immunology , Acquired Immunodeficiency Syndrome/blood , Acquired Immunodeficiency Syndrome/virology , Animals , Base Sequence , DNA Primers , DNA, Viral/genetics , Giant Cells , HIV-1/classification , HIV-1/genetics , Immunoblotting , Male , Molecular Sequence Data , Pan troglodytes , Polymerase Chain Reaction/methods , Species Specificity , Time Factors
14.
J Infect Dis ; 172(1): 250-3, 1995 Jul.
Article in English | MEDLINE | ID: mdl-7797924

ABSTRACT

A Vero cell line was investigated as a suitable host system for primary isolation and cultivation of influenza A viruses. The efficiency of primary isolation for currently circulating (H3N2) strains was similar in Vero and MDCK cells. Of 72 egg-adapted strains investigated, 90.3% were detectable hy hemagglutinin (HA) titration in Vero cells after the first passage and 51.4% after the second. The amino acid sequences of the HA1 region of influenza A viruses isolated and passaged in Vero cells were identical to those of their MDCK-grown counterparts. At low MOI, high yields of influenza virus were achieved in Vero cells by multiple additions of trypsin to the medium. After 20 passages of A/England/1/53 (H1N1) in Vero cells, the titer of infectious virus was 8.37 log10 TCID50/mL, and virus protein yields were as high as in MDCK cells.


Subject(s)
Influenza A virus/physiology , Virus Replication , Amino Acid Sequence , Animals , Base Sequence , Cell Line , Child , Chlorocebus aethiops , Dogs , Hemagglutinin Glycoproteins, Influenza Virus , Hemagglutinins, Viral/analysis , Hemagglutinins, Viral/chemistry , Humans , Influenza A virus/isolation & purification , Influenza, Human/virology , Kidney , Polymerase Chain Reaction , RNA, Viral/analysis , Vero Cells , Viral Envelope Proteins/analysis , Viral Plaque Assay
15.
Vaccine ; 13(6): 539-49, 1995 Apr.
Article in English | MEDLINE | ID: mdl-7483774

ABSTRACT

A canarypox-based (ALVAC) recombinant expressing the rabies G glycoprotein has been utilized to assess in vitro and in vivo biological properties of the canarypox virus vector system. In vitro studies have shown that no replication of the virus can be detected on six human-derived cell lines, nor can the virus be readily adapted to replicate on non-avian cells. Expression of the rabies G can be detected on all cell lines analyzed in the absence of productive viral replication. Analysis of viral-specific DNA accumulation indicated that the block in the replication cycle in the human cell lines analyzed occurred prior to DNA replication. The exact nature of the block, however, remains unknown. The concept of using a non-replicating immunization vehicle has been demonstrated through extensive in vivo studies in a range of species including non-human primates and humans. The results of such in vivo studies have exemplified the safety and immunogenicity of the ALVAC vaccine vector.


Subject(s)
Antigens, Viral , Avipoxvirus/immunology , Canaries/virology , Rabies Vaccines/immunology , Vaccines, Synthetic/immunology , Viral Vaccines/immunology , Animals , Cells, Cultured , Chlorocebus aethiops , DNA, Viral/genetics , DNA, Viral/metabolism , Gene Expression , Genetic Vectors , Glycoproteins/genetics , Glycoproteins/immunology , Guinea Pigs , Humans , Macaca mulatta , Mice , Pan troglodytes , Rabbits , Rabies Vaccines/adverse effects , Rabies Vaccines/genetics , Sensitivity and Specificity , Vaccines, Synthetic/adverse effects , Vaccines, Synthetic/genetics , Vero Cells , Viral Envelope Proteins/genetics , Viral Envelope Proteins/immunology , Viral Vaccines/adverse effects , Viral Vaccines/genetics
16.
AIDS Res Hum Retroviruses ; 11(3): 373-81, 1995 Mar.
Article in English | MEDLINE | ID: mdl-7598771

ABSTRACT

The safety and the immunogenicity of a recombinant canarypox live vector expressing the human immunodeficiency virus type 1 (HIV-1) gp160 gene from the MN isolate, ALVAC-HIV (vCP125), followed by booster injections of a soluble recombinant hybrid envelope glycoprotein MN/LAI (rgp160), were evaluated in vaccinia-immune, healthy adults at low risk for acquiring HIV-1 infection. Volunteers (n = 20) received vCP125 (10(6) TCID50) at 0 and 1 month, followed randomly by rgp160 formulated in alum or in Freund's incomplete adjuvant (FIA) at 3 and 6 months. Local and systemic reactions were mild or moderate and resolved within the first 72 hr after immunization. No significant biological changes in routine tests were observed in any volunteer. Two injections of vCP125 did not elicit antibodies. Neutralizing antibodies (NA) against the HIV-1 MN isolate were detected in 65 and 90% of the subjects after the first and the second rgp 160 booster injections, respectively. Six months after the last boost, only 55% were still positive. Seven of 14 sera with the highest NA titers against MN weakly cross-neutralized the HIV-1 SF2 isolate; none had NA against the HIV-1 LAI or against a North American primary isolate. Specific lymphocyte T cell proliferation to rgp 160 was detected in 25% of the subjects after vCP125 and in all subjects after the first booster injection and 12 months after the first injection. An envelope-specific cytotoxic lymphocyte activity was found in 39% of the volunteers and characterized for some of them as CD3+, CD8+, MHC class I restricted. The adjuvant formulation did not influence significantly the immune responses.(ABSTRACT TRUNCATED AT 250 WORDS)


Subject(s)
AIDS Vaccines/therapeutic use , Acquired Immunodeficiency Syndrome/prevention & control , Gene Products, env/immunology , HIV Antibodies/blood , HIV Infections/prevention & control , HIV Seronegativity , Immunization, Secondary , Protein Precursors/immunology , Vaccines, Synthetic/therapeutic use , AIDS Vaccines/adverse effects , Acquired Immunodeficiency Syndrome/immunology , Adult , Animals , Avipoxvirus/immunology , CD4 Lymphocyte Count , CD4-CD8 Ratio , CD8-Positive T-Lymphocytes/immunology , Canaries , Enzyme-Linked Immunosorbent Assay , Female , Gene Products, env/adverse effects , HIV Envelope Protein gp160 , HIV Infections/immunology , Humans , Lymphocyte Count , Male , Middle Aged , Protein Precursors/adverse effects , Time Factors , Vaccines, Synthetic/adverse effects
17.
Dev Biol Stand ; 84: 159-63, 1995.
Article in English | MEDLINE | ID: mdl-7796949

ABSTRACT

Three highly attenuated and efficacious poxvirus-based vectors, NYVAC, ALVAC and TROVAC, are available for targeted applications as recombinant vaccines in both human and veterinary medicine. The attenuated phenotype of the three vectors is consistent with safe use for vaccination purposes, for the vaccinee, for unvaccinated contacts, and for introduction into the environment. The precise deletion of virulence and host range genes in the NYVAC vector precludes reversion to the virulent phenotype by back mutation. Dissemination of recombinant vaccines based on the NYVAC, ALVAC and TROVAC vectors is highly diminished, because of the genetic engineering in NYVAC and the natural attenuated phenotype of ALVAC and TROVAC. Studies have demonstrated that these recombinant vectors are genetically and phenotypically stable after serial passage in vitro as well as in vivo. NYVAC, ALVAC and TROVAC vectors are the only three poxvirus-based vectors that are classified as BSL1 agents.


Subject(s)
Genetic Vectors , Poxviridae/genetics , Animals , Avipoxvirus/genetics , Fowlpox virus/genetics , Humans , Safety , Vaccines, Attenuated/genetics , Vaccines, Attenuated/isolation & purification , Vaccines, Attenuated/standards , Vaccines, Synthetic/genetics , Vaccines, Synthetic/isolation & purification , Vaccines, Synthetic/standards , Vaccinia virus/genetics , Viral Vaccines/genetics , Viral Vaccines/isolation & purification , Viral Vaccines/standards
18.
Dev Biol Stand ; 84: 165-70, 1995.
Article in English | MEDLINE | ID: mdl-7796950

ABSTRACT

ALVAC recombinants have been administered to humans and animals by parenteral and oral routes without giving signs of replication, systemic dissemination or severe reaction. In principle, it should be impossible for canarypox recombinants to disseminate in the environment as they would not be synthesised in mammalian cells as complete virus. Canarypox vectors have been safe for humans, in whom there has been no evidence of replication, but more work needs to be done to prove absence of replication. Recombinants are immunogenic by the intramuscular and subcutaneous routes. They are also immunogenic when given orally, but the dose required is still under study. Canarypox recombinants effectively prime the immune system for induction of antibodies and CD8 cell-mediated cytotoxicity by protein antigens. Antibody responses are not influenced by prior inoculation of canarypox, of subunit vaccine corresponding to the gene insert, or of vaccinia. Canarypox virus is attenuated for canaries, in which species it is already widely used. In principle, it is non-infectious for humans or other mammals. It may be infectious for other birds.


Subject(s)
Avipoxvirus/genetics , Genetic Vectors/adverse effects , Vaccines, Synthetic/adverse effects , Vaccines, Synthetic/genetics , AIDS Vaccines/administration & dosage , AIDS Vaccines/genetics , AIDS Vaccines/pharmacology , Animals , Avipoxvirus/physiology , Genetic Vectors/pharmacology , Humans , Rabies Vaccines/adverse effects , Rabies Vaccines/genetics , Rabies Vaccines/pharmacology , Safety , Vaccines, Attenuated/adverse effects , Vaccines, Attenuated/genetics , Vaccines, Attenuated/pharmacology , Vaccines, Synthetic/pharmacology , Viral Vaccines/adverse effects , Viral Vaccines/genetics , Viral Vaccines/pharmacology , Virus Replication
19.
Lab Invest ; 71(3): 350-8, 1994 Sep.
Article in English | MEDLINE | ID: mdl-7933985

ABSTRACT

BACKGROUND: Adenovirus type-5 (Ad5) recombinant viruses with replacement of the 1.9 kb XbaI fragment in the early region 3 (E3) by foreign genes have been constructed with the ultimate goal of inducing immune responses to the product of the inserted gene against a variety of virus infections. The pathogenicity of these recombinants, however, has not been studied. EXPERIMENTAL DESIGN: Histopathologic changes induced in cotton rat and mouse lung by E3-replacement-Ad5 recombinant or wild-type Ad (Wt-Ad) or E3-deleted mutant (Ad5-delta E3) viruses were compared. Expression of viral mRNA and replication of these viruses in cotton rat and mouse lungs, as well as in human tissue culture cells, were assayed. Expression of class I major histocompatibility complex antigens and the E3-14.7 kilodalton protein in virus-infected cells were also analyzed. RESULTS: An Ad5 recombinant, Ad-human cytomegalovirus glycoprotein B (Ad-HCMV.gB), in which the E3 region is replaced by the full-length gB gene of HCMV and with a genome size exceeding that of Wt-Ad, induced mild histopathologic responses in cotton rat and mouse lungs, comparable with those of Wt-Ad, but less severe than those of Ad5-delta E3. Analysis indicated that neither class I major histocompatibility complex expression on the cell surface nor differential expression of the protective E3-14.7 kilodalton protein underlies the pathologic differences observed in cells infected with Ad5-delta E3 or the Ad-HCMV.gB recombinant. In the mouse lung, another Ad-E3 replacement recombinant, Ad-herpes simplex glycoprotein B (HSV.gB), containing the complete HSV.gB gene and with a genome size larger than that of Wt-Ad, also induced a very mild inflammatory response. However, two recombinants with truncated forms of the HCMV.gB (Ad-HCMV.gB.155) or HSV.gB genes (Ad-HSV.gB.147) produced more severe histopathologic changes than the Wt-Ad or the recombinants with the full complement of HCMV.gB or HSV.gB genes. Ad5 and some of the recombinants replicated in mouse and cotton rat lung, and the extent of replication was inversely proportional to genome size, both in the lung and in human tissue culture cells. Infectious virus titers were, however, higher in cotton rat than in mouse lung. In situ hybridization analysis of cotton rat and mouse lung infected with Wt-Ad, Ad5-delta E3, or Ad-HCMV.gB virus revealed expression of Ad early/late mRNA predominantly in bronchial epithelial cells. CONCLUSIONS: These data not only confirm that E3-deleted viruses induce more severe pathologic changes in cotton rat lungs than Wt-Ad viruses (Ginsberg et al., Proc Natl Acad Sci USA 1989;86:3823-7) but led to the observation that some E3 replacement recombinants also lacking the expression of the 19 and 14.7 kilodalton proteins are significantly less pathogenic in cotton rats and mice than an E3-deleted virus. Pathogenicity and replication of the recombinant viruses inversely correlate with the genomic size.


Subject(s)
Adenoviridae/genetics , Adenoviridae/pathogenicity , Gene Deletion , Recombination, Genetic , Adenoviridae Infections/pathology , Animals , Bronchi/metabolism , Histocompatibility Antigens Class I/metabolism , Humans , Lung/pathology , Mice , Mice, Inbred BALB C , Mice, Inbred CBA , Mutation , RNA, Messenger/metabolism , RNA, Viral/metabolism , Sigmodontinae , Viral Proteins/metabolism , Virus Replication
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