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Gallo Progress Report Abstracts continued 

 
Submitted September 8, 2011

Our program tests the hypothesis that antibodies to epitopes associated with the co-receptor binding domain that is exposed on the HIV-1 envelope glycoprotein consequent to CD4 binding (CD4i epitopes) can mediate sterilizing protection against HIV-1. Two active immunization projects are underway to test this hypothesis.

First, we are optimizing a parenteral DNA-prime, protein-boost protocol in rhesus macaques to elicit high titers of CD4i antibodies. We have primed rhesus macaques with DNA vaccines encoding single chain protein (rhFLSC) in which gp120 of the HIV-1Ba-L isolate is fused with the outer two domains of rhesus CD4 that selectively elicits antibody responses to CD4i epitopes. In two independent studies, rhFLSC elicits non-sterilizing protection against a rectal challenge with SHIV162P3. A key element of optimizing the DNA-prime, protein-boost strategy is the choice of adjuvant for the protein boost. Consequent to our annual review, we established a collaboration with investigators at the Infectious Disease Research Institute (IDRI) who provided an optimal adjuvant for our studies. We have carried out immunization studies, including longevity analysis of the antibody response elicited by rhFLSC. These studies showed that while rhFLSC is immunogenic as a subunit protein in the IDRI adjuvant, DNA priming elicits more potent neutralizing antibody responses. This information will be used in the last two years of our program to design an challenge study in non-human primates to determine if sterilizing protection can be elicited.

Second, based on results obtained in the first phase of our program we initiated the development of a vesicular stomatitis virus (VSV) mucosal vaccine protocol to elicit mucosal immunity to rhFLSC. This tack was taken because of poor immunogenicity of rhFLSC when administered with cholera toxin in our original program. We have developed the initial fusions of rhFLSC with the Gstem transmembrane protein of VSV and obtained expression of the rhFLSC-Gstem chimera on the surface of attenuated VSV. Studies are underway to completely characterize the chimeric protein and to establish its immunogenicity in rodents as a prelude to carrying out a mucosal challenge study in non-human primates in the last year of our program.

We are also testing the above hypothesis by passive immunization using both human and rhesus macaque monoclonal antibodies (mAbs) specific for CD4i epitopes. We have developed new methods to census memory B cells (BMem) in humans and rhesus macaques for reactivity with specific Env epitopes and to capture the desired reactivities as mAbs. We have isolated a large panel of mAbs specific for CD4i epitopes and have defined three specificity groups based on the degree of binding enhancement by CD4. Some of these mAbs are neutralizing some are not. Most importantly, we have identified new mAbs that recognize only Env-CD4 complexes. These mAbs are non-neutralizing but mediate potent ADCC on target cells sensitized with gp120, trimeric gp140, or pseudovirions. We have obtained the crystal structure of a prototype mAb of this type and have indications that it recognizes a novel CD4i epitope that are exposed only during viral entry. This mAb, along with others are candidates for the passive immunization studies to be carried out in the last two years of our program.

Submitted January 11, 2011 (Interim Report)

Our program remains on track to test the hypothesis that antibodies to epitopes associated with the co-receptor binding domain that is exposed on the HIV-1 envelope glycoprotein consequent to CD4 binding (CD4i epitopes) can mediate sterilizing protection against HIV-1. Two active immunization projects are underway to test this hypothesis.

In the first project, we have optimized a parenteral DNA-prime, protein-boost protocol in rhesus macaques to elicit high titers of CD4i antibodies. We primed rhesus macaques with DNA vaccines encoding single chain protein (rhFLSC) in which gp120 of the HIV-1 Ba-L isolate is fused with the outer two domains ofrhesus CD4 that selectively elicits antibody responses to CD4i epitopes.In two independent studies, rhFLSC elicits non-sterilizing protection against a rectal challenge with: A key element of optimizing the DNA-prime, protein-boost strategy is the choice of adjuvant for the protein boost. We established a collaboration with investigators at the Infectious Disease Research Institute (IDRI) who provided an optimal adjuvant for our studies. We have developed data indicating that while the IDRI adjuvant can increase immunogenicity of rhFLSC without DNA priming, there is an effect of including the DNA immunization on both magnitude of the neutralizing antibody response and possibly duration of the response. We are on track for using this information to configure a DNA-prime, protein-boost study to determine whether we get sterilizing protection against SHIV162p3.

In the second project, we are optimizing a mucosal immunization protocol using rhFLSC as the immunogen.As noted previously, we altered our original approach to more selectively target rhFLSC to mucosal surfaces by expressing it on the surface vesicular stomatitis virus (VSV), which is a potent mucosal vaccine vector.We have developed a VSV-rhFLSC vector that stably expresses rhFLSC on the virion surface and it is under evaluation in guinea pigs. We anticipate having the data in hand within the next two months.If the studies proceed as predicted and the construct is immunogenic in guinea pigs, we will use it to carry out the propose SHIV162p3 challenge study.In the third project, we are directly testing above hypothesis by passive immunization using both human and rhesus macaque monoclonal antibodies (mAbs) specific for CD4i epitopes.We have developed a comprehensive panel of human and rhesus macaque CD4i mAbs that can be categorized into three groups based on the relative mAb reactivities with gp120-CD4 complexes as opposed to gp120 alone. CD4i-I mAbs exhibit approximately a 10-fold to 30-fold preference for the complexes, CD4i-II mAbs exhibit approximately a 100-fold to 1000-fold preference for the complexes, and CD4i-III mAbs are strictly specific for the complexes with no detectable reactivity for free gp120.Seventy five percent of the CD4i-II group are neutralizing where we see selection for increasing neutralization breadth and potency as a function of somatic hypermutation and binding to a V3-core structure.We have obtained the crystal structure of one mAb that exhibits neutralization breadth and its structural features are consistent with strong selection in vivo. By contrast, none of the CD4i-I mAbs and approximately fifty percent of the CD4i-III mAbs were neutralizing. Among the neutralizing mAbs in the CD4i-III group, potency and breadth were modest and there was no obvious selection for increasing breadth as a function of somatic hypermutation. Most important, all groups had mAbs that exhibit significant ADCC in the absence of neutralization. The most potent mAbs are in the CD4i-I and CD4i-III groups where the CD4i-I mAbs most closely resemble A32 and C11. By contrast, we have a uniquely potent CD4i-III mAb (mAb N12-I15) that recognizes a new epitope involving the co-receptor and V1/V2 regions. In the CD4i-III mAbs we see co-selection for ADCC potency and short CDR-H3 segments. This is confirmed in the crystal structure of N12-I15, which has an unusual paratope that is trough like. We have also adopted systems to evaluate the binding of the mAbs to either free virions in solution (Fluorescence Correlation Spectroscopy; FCS) or to live target cells in a dynamic setting (live cell confocal microscopy). We find that all anti-CD4i Mabs demonstrate the expected CD4-dependent binding to envelope on free virions saturated with soluble CD4. However, we are observing that cell surface-bound virions seem to globally present CD4i-I epitopes on their envelope spikes even when distal to the target cell surface. This exposure pattern is consistent with our observations of ADCC activity mediated by anti-CD4i Mabs against viral particles. Taken together, this information puts us in an excellent position to carry out highly informed passive immunizations with lead mAbs from each of the anti-CD4i groups. These studies are on target for the next eighteen months of our program.

Submitted August 30, 2010

Our program tests the hypothesis that antibodies to epitopes associated with the co-receptor binding domain that is exposed on the HIV-1 envelope glycoprotein consequent to CD4 binding (CD4i epitopes) can mediate sterilizing protection against HIV-1. Two active immunization projects are underway to test this hypothesis.

First, we are optimizing a parenteral DNA-prime, protein-boost protocol in rhesus macaques to elicit high titers of CD4i antibodies.  We have primed rhesus macaques with DNA vaccines encoding single chain protein (rhFLSC) in which gp120 of the HIV-1Ba-L  isolate is fused with the outer two domains of rhesus CD4 that selectively elicits antibody responses to CD4i epitopes.  In two independent studies, rhFLSC elicits non-sterilizing protection against a rectal challenge with SHIV162P3.  A key element of optimizing the DNA-prime, protein-boost strategy is the choice of adjuvant for the protein boost.  Consequent to our annual review, we established a collaboration with investigators at the Infectious Disease Research Institute (IDRI) who provided an optimal adjuvant for our studies.  We have carried out immunization studies, including longevity analysis of the antibody response elicited by rhFLSC.  These studies showed that while rhFLSC is immunogenic as a subunit protein in the IDRI adjuvant, DNA priming elicits more potent neutralizing antibody responses.  This information will be used in the last two years of our program to design a challenge study in non-human primates to determine if sterilizing protection can be elicited.

Second, based on results obtained in the first phase of our program we initiated the development of a vesicular stomatitis virus (VSV) mucosal vaccine protocol to elicit mucosal immunity to rhFLSC.  This tack was taken because of poor immunogenicity of rhFLSC when administered with cholera toxin in our original program.  We have developed the initial fusions of rhFLSC with the Gstem transmembrane protein of VSV and obtained expression of the rhFLSC-Gstem chimera on the surface of attenuated VSV. Studies are underway to completely characterize the chimeric protein and to establish its immunogenicity in rodents as a prelude to carrying out a mucosal challenge study in non-human primates in the last year of our program.

We are also testing the above hypothesis by passive immunization using both human and rhesus macaque monoclonal antibodies (mAbs) specific for CD4i epitopes.  We have developed new methods to census memory B cells (BMem) in humans and rhesus macaques for reactivity with specific Env epitopes and to capture the desired reactivities as mAbs.  We have isolated a large panel of mAbs specific for CD4i epitopes and have defined three specificity groups based on the degree of binding enhancement by CD4.  Some of these mAbs are neutralizing while some are not.  Most importantly, we have identified new mAbs that recognize only Env-CD4 complexes.  These mAbs are non-neutralizing but mediate potent ADCC on target cells sensitized with gp120, trimeric gp140, or pseudovirions.  We have obtained the crystal structure of a prototype mAb of this type and have indications that it recognizes a novel CD4i epitope that are exposed only during viral entry.  This mAb, along with others, are candidates for the passive immunization studies to be carried out in the last two years of our program. 

 
Submitted April 15, 2010 (Interim Report)

Our program continues to the hypothesis that antibodies to epitopes associated with the co-receptor binding domain that is exposed on the HIV-1 envelope glycoprotein consequent to CD4 binding (CD4i epitopes) can mediate sterilizing protection against HIV-1. Two active immunization projects are underway to test this hypothesis.

In the first project, we are optimizing a parenteral DNA-prime, protein-boost protocol in rhesus macaques to elicit high titers of CD4i antibodies.   We have primed rhesus macaques with DNA vaccines encoding single chain protein (rhFLSC) in which gp120 of the HIV-1Ba-L  isolate is fused with the outer two domains of  rhesus CD4 that  selectively elicits antibody responses to CD4i epitopes.  In two independent studies, rhFLSC elicits non-sterilizing protection against a rectal challenge with SHIV162P3.  A key element of optimizing the DNA-prime, protein-boost strategy is the choice of adjuvant for the protein boost. We established a collaboration with investigators at the Infectious Disease Research Institute (IDRI) who provided an optimal adjuvant for our studies.  We have obtained boosting with the adjuvant and developed new data that its use might supplant the need for DNA priming.  If so, this would greatly simplify the immunization scheme.  Studies are underway to determine the longevity of the antibody responses elicited by immunization with rhFLSC as a protein only vaccine using this adjuvant.   These results will be compared with those obtained by a DNA-prime, rhFSLC protein-boost protocol to design a protection study to be carried out in the next years of our program.

In the second project, we are optimizing a mucosal immunization protocol using rhFLSC as the immunogen.  Based on the unexpectedly poor mucosal immunogenicity of rhFSLC when delivered with cholera-toxin, we have altered our original approach in order to to more selectively target rhFLSC to mucosal surfaces by expressing it on the surface vesicular stomatitis virus (VSV), which is a potent mucosal vaccine vector.  This alteration was approved after our annual review and the rhFSLC-VSV vaccine is under development.  We anticipate having it in hand and the necessary small animal immunogenicity studies carried out as defined in our modified schedule to set the stage for a protection study to be carried out in the next years of our program.

In the third project, we are directly testing above hypothesis by passive immunization using both human and rhesus macaque monoclonal antibodies (mAbs) specific for CD4i epitopes.  We have developed new methods to census memory B cells (BMem) for reactivity with specific Env epitopes and to capture the desired reactivities as mAbs.  We are isolating a large panel of CD4i mAbs to obtain a detailed picture of the fine specificities that populate that anti-CD4i antibody response and, of equal importance, to determine if there is an association between specificity and effector function.  We have a number of candidate mAbs in hand where there appears to be such a relationship.  The goal is to develop a mAb panel for passive immunization against a mucosal challenge with SHIV162P3 in the last two years of our program.

 
Submitted August 27, 2009

Our program tests the hypothesis that antibodies to epitopes associated with the co-receptor binding domain that is exposed on the HIV-1 envelope glycoprotein consequent to CD4 binding (CD4i epitopes) can mediate sterilizing protection against HIV-1. Two active immunization projects are underway to test this hypothesis.

First, we are optimizing a parenteral DNA-prime, protein-boost protocol in rhesus macaques to elicit high titers of CD4i antibodies.   We have primed rhesus macaques with DNA vaccines encoding single chain protein (rhFLSC) in which gp120 of the HIV-1Ba-L  isolate is fused with the outer two domains of  rhesus CD4 that  selectively elicits antibody responses to CD4i epitopes.  In two independent studies, rhFLSC elicits non-sterilizing protection against a rectal challenge with SHIV162P3.  A key element of optimizing the DNA-prime, protein-boost strategy is the choice of adjuvant for the protein boost.  Consequent to our annual review, we established a collaboration with investigators at the Infectious Disease Research Institute (IDRI) who provided an optimal adjuvant for our studies.  We have completed the immunization studies and are now accumulating and analyzing the remaining immunogenicity data. 

Second, we are optimizing a mucosal immunization protocol using rhFLSC as the immunogen and wild-type CT (CT) as the adjuvant.  We have completed the first series of immunizations and found an appropriate dose of CT to use as a mucosal adjuvant for rectal immunizations, as indicated by clear-cut titration of the anti-CT antibody response.   However, the antibody responses to rhFLSC were unexpectedly variable and did not titrate with CT dose.  We have repeated the serological analyses using more sensitive techniques, again, confirming the variable nature of the responses. Based on these results we have developed an alternative plan that uses attenuated Vesicular Stomatitis Virus (VSV) as a mucosal vaccine vector for rhFLSC.

We are also testing the above hypothesis by passive immunization using both human and rhesus macaque monoclonal antibodies (mAbs) specific for CD4i epitopes.  We have developed new methods to census memory B cells (BMem) for reactivity with specific Env epitopes and to capture the desired reactivities as mAbs.  We are isolating a large panel of CD4i mAbs to obtain a detailed picture of the fine specificities that populate that anti-CD4i antibody response and, of equal importance, to determine if there is an association between specificity and effector function.  We have a number of candidate mAbs in hand where there appears to be such a relationship. We have identified several mAbs that are prime candidates to carry out passive immunization studies in the next years of our program.

 
Submitted February 2, 2009 (Interim Report)

Our program tests the hypothesis that antibodies to epitopes associated with the co-receptor binding domain that is exposed on the HIV-1 envelope glycoprotein consequent to CD4 binding (CD4i epitopes) can mediate sterilizing protection against HIV-1. Two active immunization projects are underway to test this hypothesis.

First, we are optimizing a parenteral DNA-prime, protein-boost protocol in rhesus macaques to elicit high titers of CD4i antibodies.   We have primed rhesus macaques with DNA vaccines encoding single chain protein (rhFLSC) in which gp120 of the HIV-1Ba-L  isolate is fused with the outer two domains of  rhesus CD4 that  selectively elicits antibody responses to CD4i epitopes.  In two independent studies, rhFLSC elicits non-sterilizing protection against a rectal challenge with SHIV162P3.  A key element of optimizing the DNA-prime, protein-boost strategy is the choice of adjuvant for the protein boost.  Consequent to our annual review, we established a collaboration with investigators at the Infectious Disease Research Institute (IDRI) who provided an optimal adjuvant for our studies.  The adjuvant is in hand and will be used to boost the primed macaques in the first week of February 2009.  We anticipate that this adjuvant will enhance the immunogenicity of rhFLSC substantially over the original adjuvant proposed in our studies.

Second, we are optimizing a mucosal immunization protocol using rhFLSC as the immunogen and wild-type cholera toxin (CT) as the adjuvant.  We have completed the first series of immunizations and found an appropriate dose of CT to use as a mucosal adjuvant for rectal immunizations, as indicated by clear-cut titration of the anti-CT antibody response.   However, the antibody responses to rhFLSC were unexpectedly variable and did not titrate with CT dose.  We are examining the basis for this effect before carrying out additional dose titration studies of rhFLSC in the second phase of this study.  

We are also testing the above hypothesis by passive immunization using both human and rhesus macaque monoclonal antibodies (mAbs) specific for CD4i epitopes.  We have developed new methods to census memory B cells (BMem) for reactivity with specific Env epitopes and to capture the desired reactivities as mAbs.  We are isolating a large panel of CD4i mAbs  to obtain a detailed picture of the fine specificities that populate that anti-CD4i antibody response and, of equal importance, to determine if there is an association between specificity and effector function.  We have a number of candidate mAbs in hand where there appears to be such a relationship.  The goal is to develop a mAb panel for passive immunization against a mucosal challenge with SHIV162P3 in the last two years of our program. 

 

Submitted July 1, 2008

This VDAC will test the hypothesis that immunization with conformationally constrained gp120-CD4 vaccines elicits sterilizing immunity against rectal challenge with SHIV.  Two immunization studies are underway in rhesus macaques that are the first steps in optimizing systemic and mucosal immunization protocols to enhance immune responses to levels predicted to be required for sterilizing protection.  These studies led by Dr. Tony Devico and David Pauza, respectively, and are on target as originally proposed. Data from these studies will become available in the third and fourth quarters of 2008.  A third objective, led by Dr. George K. Lewis, uses passive immunization with monoclonal antibodies specific for novel gp120-CD4 epitopes (CD4i epitopes) of the vaccine to determine whether sterilizing immunity is possible for CD4i epitopes.  Significant progress has been made in that a new algorithm for rapidly generating full-length human monoclonal antibodies has been developed and used to isolate 16 new monoclonal antibodies (mAbs) that recognize CD4i epitopes with many more under development.   The mAbs are being analyzed for epitope specificity, neutralization breadth, and the ability to mediate Fc-dependent effector functions such as antibody-dependent cell mediated cytotoxicity. This method is being extended to rhesus macaques immunized with gp120-CD4 immunogens or infected with a model mucosal SHIV virus.  In summary, year 1 of our VDAC is on schedule and new data are already starting to emerge that will permit testing our principal hypothesis that immunization with conformationally constrained gp120-CD4 vaccines elicits sterilizing immunity.

 
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