Two commercially available expression vectors were modified to generate plasmids pGEXcPk and pQ9c... more Two commercially available expression vectors were modified to generate plasmids pGEXcPk and pQ9cPk. Proteins expressed from pGEXcPk and pQ9cPk had a short oligopeptide tag termed Pk at their carboxy termini and either glutathione S-transferase (GST) or a small histidine (His) tag, respectively, at their N termini. GST fusion proteins can be purified on immobilized glutathione and proteins coupled to the His tag selectively bind to Ni(2+)-NTA columns. The Pk tag is recognized by monoclonal antibody (MAb) SV5-P-k, previously produced in our laboratory. Thus proteins expressed from the pGEXcPk and pQ9cPk vectors can be purified in a two-step procedure, first via the N-terminal tag and second via the C-terminal tag. The combination of two affinity purification steps significantly improves the antigen purity and selects for full-size proteins. Moreover, by using the MAbSV5-P-k in the second purification step, Pk-linked antigens can be assembled directly into solid matrix-antibody-antigen (SMAA) complexes for use as vaccines. The genes for nef, endonuclease, p15, p17, p27, protease, Rev, reverse transcriptase (rt), tat, vif, vpr, and vpx of simian immunodeficiency virus (SIV mac 251) were cloned and expressed as both GST-SIV-Pk and His-SIV-Pk proteins. Multivalent SMAA complexes were made that contained His-p17-Pk, His-p27-Pk, His-rt-Pk, His-vpx-Pk, and His-vpr-Pk. Following two immunizations of mice with this mixture, antibodies could be detected to all five SIV antigens. When compared to single-protein immunizations, the immunogenicity of some of the proteins in this cocktail was either enhanced or decreased. Mice were also immunized with His-p17-Pk or His-p17-Pk-antibody complexes in the presence or absence of alum. The antibody-antigen complexes induced two- to four-fold higher antibody levels than antigen alone but did not appear to be more immunogenic in inducing lymphoproliferative responses. Sera from SIV-infected macaques were tested for the presence of antibodies reacting with the recombinant proteins by Western blot analysis. Antibodies to endonuclease, p15, p17, p27, rt, and vif were readily detected, antibodies against protease and vpx were present at much lower levels, but no antibodies were detected to nef, rev, tat, or vpr. Thus, we have developed a comprehensive range of reagents (available on request) that can be used to examine immune responses to SIV in both mice and monkeys.
Generating a protective immune response to viral infection is known to depend upon the priming an... more Generating a protective immune response to viral infection is known to depend upon the priming and clonal expansion of virus-specific CD8 T cells by Ag-loaded dendritic cells (DC) within secondary lymphoid tissue. However, the actual initiation of the response involves critical upstream events that control the recruitment of mature Ag-charged DC from the periphery via afferent lymphatics, events that are
Cytotoxic T cells substantially contribute to the control of intracellular pathogens such as huma... more Cytotoxic T cells substantially contribute to the control of intracellular pathogens such as human immunodeficiency virus type 1 (HIV-1). Here, we evaluated the immunopeptidome of Jurkat cells infected with the vaccine candidate MVA.HIVconsv which delivers HIV-1 conserved antigenic regions using modified vaccinia Ankara (MVA). We employed liquid chromatography tandem mass spectrometry (LC-MS/MS) to identify 6,358 unique peptides associated with class I human leukocyte antigen (HLA), of which 98 peptides were derived from the MVA vector and 7 from the HIVconsv immunogen. Human vaccine recipients responded to the peptide sequences identified by LC-MS/MS. Peptides derived from the conserved HIV-1 regions were readily detected as early as 1.5 hour after the MVA.HIVconsv infection. Four of the 7 conserved peptides were followed between 0 and 3.5 hours of infection using quantitative mass spectrometry (Q-MS) and their abundance in HLA class I association reflected levels of the whole HIVc...
IAVI-006 was the first large randomised, double-blinded, placebo-controlled Phase I clinical tria... more IAVI-006 was the first large randomised, double-blinded, placebo-controlled Phase I clinical trial to systematically investigate the prime-boost strategy for induction of HIV-1 specific CD8+ cytotoxic T-lymphocytes (CTL) in a factorial trial design using (i) priming with 0.5 mg or 2 mg of pTHr.HIVA DNA vaccine, followed by (ii) two booster vaccinations with 5 x 10(7) MVA.HIVA at weeks 8 and 12 (early boost) or weeks 20 and 24 (late boost). This study set the basis for later clinical trials and demonstrated the safety of these candidate HIV vaccines. The safety and immunogenicity results are presented and the lessons derived from this clinical trial are discussed.
DNA and modified vaccinia virus Ankara (MVA) are vaccine vehicles suitable and safe for use in hu... more DNA and modified vaccinia virus Ankara (MVA) are vaccine vehicles suitable and safe for use in humans. Here, by using a multicytotoxic T-lymphocyte (CTL) epitope gene and a DNA prime-MVA boost vaccination regimen, high levels of CTLs specific for a single simian immunodeficiency virus (SIV) gag-derived epitope were elicited in rhesus macaques. These vaccine-induced CTLs were capable of killing SIV-infected cells in vitro. Fluorescence-activated cell sorter analysis using soluble tetrameric major histocompatibility complex-peptide complexes showed that the vaccinated animals had 1 to 5% circulating CD8(+) lymphocytes specific for the vaccine epitope, frequencies comparable to those in SIV-infected monkeys. Upon intrarectal challenge with pathogenic SIVmac251, no evidence for protection was observed in at least two of the three vaccinated animals. This study does not attempt to define correlates of protective immunity nor design a protective vaccine against immunodeficiency viruses, b...
Development of an HIV vaccine presents a formidable challenge. One of the unresolved, yet central... more Development of an HIV vaccine presents a formidable challenge. One of the unresolved, yet central issues is the importance of HIV variability. Here we argue that even with the recent focus on the induction of T cell-mediated immunity, HIV vaccines should match the local circulating HIV clades. Whether used alone or in a combination with vaccines eliciting HIV-neutralizing antibodies, efforts must be made to develop a T cell vaccine that stimulates a broad and long-lasting response.
Most children in Africa receive their vaccine against tuberculosis at birth. Those infants born t... more Most children in Africa receive their vaccine against tuberculosis at birth. Those infants born to human immunodeficiency virus type 1 (HIV-1)-positive mothers are at high risk of acquiring HIV-1 infection through breastfeeding in the first weeks of their lives. Thus, the development of a vaccine which would protect newborns against both of these major global killers is a logical yet highly scientifically, ethically, and practically challenging aim. Here, a recombinant lysine auxotroph of Mycobacterium bovis bacillus Calmette-Guérin (BCG), a BCG strain that is safer than those currently used and expresses an African HIV-1 clade-derived immunogen, was generated and shown to be stable and to induce durable, high-quality HIV-1-specific CD4(+)- and CD8(+)-T-cell responses. Furthermore, when the recombinant BCG vaccine was used in a priming-boosting regimen with heterologous components, the HIV-1-specific responses provided protection against surrogate virus challenge, and the recombinan...
Cytotoxic T-lymphocyte (CTL) responsestoherpes simplex virus (HSV)polypeptides playan important r... more Cytotoxic T-lymphocyte (CTL) responsestoherpes simplex virus (HSV)polypeptides playan important role inrecoveryfrominfection andinpreventing latency. We havepreviously shownthatglycoprotein B (gB)isa majortarget recognized byHSV-specific CTLsinC57BL/6(H-2b) andBALB/c(H-2d) micebutnotinCBA/J (H-2k) mice(L.A.Witmer, K.L.Rosenthal, F.L.Graham,H.M.Friedman, A.Yee,andD.C.Johnson, J. Gen.Virol. 71:387-396, 1990). Inthis report, we utilize adenovirus vectors expressing gB withvarious deletions tolocalize an immunodominant site ingB,recognized byH-2b-restricted anti-HSV CTLs,toa region between residues 462and594.Overlapping peptides spanning this region were
BackgroundOne of the big roadblocks in development of HIV-1/AIDS vaccines is the enormous diversi... more BackgroundOne of the big roadblocks in development of HIV-1/AIDS vaccines is the enormous diversity of HIV-1, which could limit the value of any HIV-1 vaccine candidate currently under test.Methodology and FindingsTo address the HIV-1 variation, we designed a novel T cell immunogen, designated HIVCONSV, by assembling the 14 most conserved regions of the HIV-1 proteome into one chimaeric protein. Each
Eliciting neutralizing antibodies capable of inactivating a broad spectrum of HIV-1 strains is a ... more Eliciting neutralizing antibodies capable of inactivating a broad spectrum of HIV-1 strains is a major goal of HIV-1 vaccine design. The challenge is that envelopes (Envs) of circulating viruses are almost certainly different from any Env used in a vaccine. A novel immunogen composed of a highly diverse set of gp140 Envs including subtypes A, B, C, D and F was developed to stimulate a more cross-neutralizing antibody response. Env heterotrimers composed of up to 54 different gp140s were produced with the aim of focusing the response to the conserved regions of Env while reducing the dominance of any individual hypervariable region. Heterotrimeric gp140 Envs of inter- and intra-subtype combinations were shown to bind CD4 and a panel of neutralizing monoclonal antibodies with similar affinity to monovalent UG37 gp140. Macaques immunized with six groups of heterotrimer mixtures showed slightly more potent neutralizing antibody responses in TZM-BL tier 1 and A3R5 tier 2 pseudovirus assays than macaques immunized with monovalent Env gp140, and exhibited a marginally greater focus on the CD4-binding site. Carbopol enhanced neutralization when used as an adjuvant instead of RIBI in combination with UG37 gp140. These data indicate that cross-subtype heterotrimeric gp140 Envs may elicit some improvement of the neutralizing antibody response in macaques compared to monovalent gp140 Env.
We investigated whether vaccination of healthy HIV-seronegative and HIV-1-seropositive antiretrov... more We investigated whether vaccination of healthy HIV-seronegative and HIV-1-seropositive antiretroviral therapy-treated subjects with recombinant modified vaccinia virus Ankara expressing an HIV-1 immunogen (MVA.HIVA) induced MVA-specific T cell responses. Using IFN-γ Elispot assays, we observed new or increased responses to MVA virus in 52% of HIV-seronegative subjects and 93% HIV-1 seropositive subjects; MVA-specific T cell frequencies were generally low and correlated poorly with T cell responses to the HIV-1 immunogen. In two vaccinees, responses were mapped to CD8+ T cell epitopes present in replication-competent vaccinia virus. These data support further evaluation of MVA as a viral vector for HIV-1 immunogens.
Two commercially available expression vectors were modified to generate plasmids pGEXcPk and pQ9c... more Two commercially available expression vectors were modified to generate plasmids pGEXcPk and pQ9cPk. Proteins expressed from pGEXcPk and pQ9cPk had a short oligopeptide tag termed Pk at their carboxy termini and either glutathione S-transferase (GST) or a small histidine (His) tag, respectively, at their N termini. GST fusion proteins can be purified on immobilized glutathione and proteins coupled to the His tag selectively bind to Ni(2+)-NTA columns. The Pk tag is recognized by monoclonal antibody (MAb) SV5-P-k, previously produced in our laboratory. Thus proteins expressed from the pGEXcPk and pQ9cPk vectors can be purified in a two-step procedure, first via the N-terminal tag and second via the C-terminal tag. The combination of two affinity purification steps significantly improves the antigen purity and selects for full-size proteins. Moreover, by using the MAbSV5-P-k in the second purification step, Pk-linked antigens can be assembled directly into solid matrix-antibody-antigen (SMAA) complexes for use as vaccines. The genes for nef, endonuclease, p15, p17, p27, protease, Rev, reverse transcriptase (rt), tat, vif, vpr, and vpx of simian immunodeficiency virus (SIV mac 251) were cloned and expressed as both GST-SIV-Pk and His-SIV-Pk proteins. Multivalent SMAA complexes were made that contained His-p17-Pk, His-p27-Pk, His-rt-Pk, His-vpx-Pk, and His-vpr-Pk. Following two immunizations of mice with this mixture, antibodies could be detected to all five SIV antigens. When compared to single-protein immunizations, the immunogenicity of some of the proteins in this cocktail was either enhanced or decreased. Mice were also immunized with His-p17-Pk or His-p17-Pk-antibody complexes in the presence or absence of alum. The antibody-antigen complexes induced two- to four-fold higher antibody levels than antigen alone but did not appear to be more immunogenic in inducing lymphoproliferative responses. Sera from SIV-infected macaques were tested for the presence of antibodies reacting with the recombinant proteins by Western blot analysis. Antibodies to endonuclease, p15, p17, p27, rt, and vif were readily detected, antibodies against protease and vpx were present at much lower levels, but no antibodies were detected to nef, rev, tat, or vpr. Thus, we have developed a comprehensive range of reagents (available on request) that can be used to examine immune responses to SIV in both mice and monkeys.
Generating a protective immune response to viral infection is known to depend upon the priming an... more Generating a protective immune response to viral infection is known to depend upon the priming and clonal expansion of virus-specific CD8 T cells by Ag-loaded dendritic cells (DC) within secondary lymphoid tissue. However, the actual initiation of the response involves critical upstream events that control the recruitment of mature Ag-charged DC from the periphery via afferent lymphatics, events that are
Cytotoxic T cells substantially contribute to the control of intracellular pathogens such as huma... more Cytotoxic T cells substantially contribute to the control of intracellular pathogens such as human immunodeficiency virus type 1 (HIV-1). Here, we evaluated the immunopeptidome of Jurkat cells infected with the vaccine candidate MVA.HIVconsv which delivers HIV-1 conserved antigenic regions using modified vaccinia Ankara (MVA). We employed liquid chromatography tandem mass spectrometry (LC-MS/MS) to identify 6,358 unique peptides associated with class I human leukocyte antigen (HLA), of which 98 peptides were derived from the MVA vector and 7 from the HIVconsv immunogen. Human vaccine recipients responded to the peptide sequences identified by LC-MS/MS. Peptides derived from the conserved HIV-1 regions were readily detected as early as 1.5 hour after the MVA.HIVconsv infection. Four of the 7 conserved peptides were followed between 0 and 3.5 hours of infection using quantitative mass spectrometry (Q-MS) and their abundance in HLA class I association reflected levels of the whole HIVc...
IAVI-006 was the first large randomised, double-blinded, placebo-controlled Phase I clinical tria... more IAVI-006 was the first large randomised, double-blinded, placebo-controlled Phase I clinical trial to systematically investigate the prime-boost strategy for induction of HIV-1 specific CD8+ cytotoxic T-lymphocytes (CTL) in a factorial trial design using (i) priming with 0.5 mg or 2 mg of pTHr.HIVA DNA vaccine, followed by (ii) two booster vaccinations with 5 x 10(7) MVA.HIVA at weeks 8 and 12 (early boost) or weeks 20 and 24 (late boost). This study set the basis for later clinical trials and demonstrated the safety of these candidate HIV vaccines. The safety and immunogenicity results are presented and the lessons derived from this clinical trial are discussed.
DNA and modified vaccinia virus Ankara (MVA) are vaccine vehicles suitable and safe for use in hu... more DNA and modified vaccinia virus Ankara (MVA) are vaccine vehicles suitable and safe for use in humans. Here, by using a multicytotoxic T-lymphocyte (CTL) epitope gene and a DNA prime-MVA boost vaccination regimen, high levels of CTLs specific for a single simian immunodeficiency virus (SIV) gag-derived epitope were elicited in rhesus macaques. These vaccine-induced CTLs were capable of killing SIV-infected cells in vitro. Fluorescence-activated cell sorter analysis using soluble tetrameric major histocompatibility complex-peptide complexes showed that the vaccinated animals had 1 to 5% circulating CD8(+) lymphocytes specific for the vaccine epitope, frequencies comparable to those in SIV-infected monkeys. Upon intrarectal challenge with pathogenic SIVmac251, no evidence for protection was observed in at least two of the three vaccinated animals. This study does not attempt to define correlates of protective immunity nor design a protective vaccine against immunodeficiency viruses, b...
Development of an HIV vaccine presents a formidable challenge. One of the unresolved, yet central... more Development of an HIV vaccine presents a formidable challenge. One of the unresolved, yet central issues is the importance of HIV variability. Here we argue that even with the recent focus on the induction of T cell-mediated immunity, HIV vaccines should match the local circulating HIV clades. Whether used alone or in a combination with vaccines eliciting HIV-neutralizing antibodies, efforts must be made to develop a T cell vaccine that stimulates a broad and long-lasting response.
Most children in Africa receive their vaccine against tuberculosis at birth. Those infants born t... more Most children in Africa receive their vaccine against tuberculosis at birth. Those infants born to human immunodeficiency virus type 1 (HIV-1)-positive mothers are at high risk of acquiring HIV-1 infection through breastfeeding in the first weeks of their lives. Thus, the development of a vaccine which would protect newborns against both of these major global killers is a logical yet highly scientifically, ethically, and practically challenging aim. Here, a recombinant lysine auxotroph of Mycobacterium bovis bacillus Calmette-Guérin (BCG), a BCG strain that is safer than those currently used and expresses an African HIV-1 clade-derived immunogen, was generated and shown to be stable and to induce durable, high-quality HIV-1-specific CD4(+)- and CD8(+)-T-cell responses. Furthermore, when the recombinant BCG vaccine was used in a priming-boosting regimen with heterologous components, the HIV-1-specific responses provided protection against surrogate virus challenge, and the recombinan...
Cytotoxic T-lymphocyte (CTL) responsestoherpes simplex virus (HSV)polypeptides playan important r... more Cytotoxic T-lymphocyte (CTL) responsestoherpes simplex virus (HSV)polypeptides playan important role inrecoveryfrominfection andinpreventing latency. We havepreviously shownthatglycoprotein B (gB)isa majortarget recognized byHSV-specific CTLsinC57BL/6(H-2b) andBALB/c(H-2d) micebutnotinCBA/J (H-2k) mice(L.A.Witmer, K.L.Rosenthal, F.L.Graham,H.M.Friedman, A.Yee,andD.C.Johnson, J. Gen.Virol. 71:387-396, 1990). Inthis report, we utilize adenovirus vectors expressing gB withvarious deletions tolocalize an immunodominant site ingB,recognized byH-2b-restricted anti-HSV CTLs,toa region between residues 462and594.Overlapping peptides spanning this region were
BackgroundOne of the big roadblocks in development of HIV-1/AIDS vaccines is the enormous diversi... more BackgroundOne of the big roadblocks in development of HIV-1/AIDS vaccines is the enormous diversity of HIV-1, which could limit the value of any HIV-1 vaccine candidate currently under test.Methodology and FindingsTo address the HIV-1 variation, we designed a novel T cell immunogen, designated HIVCONSV, by assembling the 14 most conserved regions of the HIV-1 proteome into one chimaeric protein. Each
Eliciting neutralizing antibodies capable of inactivating a broad spectrum of HIV-1 strains is a ... more Eliciting neutralizing antibodies capable of inactivating a broad spectrum of HIV-1 strains is a major goal of HIV-1 vaccine design. The challenge is that envelopes (Envs) of circulating viruses are almost certainly different from any Env used in a vaccine. A novel immunogen composed of a highly diverse set of gp140 Envs including subtypes A, B, C, D and F was developed to stimulate a more cross-neutralizing antibody response. Env heterotrimers composed of up to 54 different gp140s were produced with the aim of focusing the response to the conserved regions of Env while reducing the dominance of any individual hypervariable region. Heterotrimeric gp140 Envs of inter- and intra-subtype combinations were shown to bind CD4 and a panel of neutralizing monoclonal antibodies with similar affinity to monovalent UG37 gp140. Macaques immunized with six groups of heterotrimer mixtures showed slightly more potent neutralizing antibody responses in TZM-BL tier 1 and A3R5 tier 2 pseudovirus assays than macaques immunized with monovalent Env gp140, and exhibited a marginally greater focus on the CD4-binding site. Carbopol enhanced neutralization when used as an adjuvant instead of RIBI in combination with UG37 gp140. These data indicate that cross-subtype heterotrimeric gp140 Envs may elicit some improvement of the neutralizing antibody response in macaques compared to monovalent gp140 Env.
We investigated whether vaccination of healthy HIV-seronegative and HIV-1-seropositive antiretrov... more We investigated whether vaccination of healthy HIV-seronegative and HIV-1-seropositive antiretroviral therapy-treated subjects with recombinant modified vaccinia virus Ankara expressing an HIV-1 immunogen (MVA.HIVA) induced MVA-specific T cell responses. Using IFN-γ Elispot assays, we observed new or increased responses to MVA virus in 52% of HIV-seronegative subjects and 93% HIV-1 seropositive subjects; MVA-specific T cell frequencies were generally low and correlated poorly with T cell responses to the HIV-1 immunogen. In two vaccinees, responses were mapped to CD8+ T cell epitopes present in replication-competent vaccinia virus. These data support further evaluation of MVA as a viral vector for HIV-1 immunogens.
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Papers by Tomás Hanke