Structural Integrity of the -Carboxyglutamic Acid Domain of Human Blood Coagulation Factor IXa Is Required for Its Binding to Cofactor VIIIa

This report describes the analysis of a novel mutant human factor IX protein from a patient with hemophilia B (factor IX activity adenine transition) in exon 2 at nucleotide 6409 which results in a glycine --> arginine substitution at amino acid 12 in the gamma-carboxyglutamic acid rich (Gla) domain of the mature protein. Factor IX was isolated by immunoaffinity chromatography from plasma obtained from the proband. The purified protein is indistinguishable from normal factor IX by polyacrylamide gel electrophoresis. Characterization of the variant in purified component assays reveals that it is activated normally by its physiologic activator factor XIa, but its phospholipid-dependent activation by the factor VIIa-tissue factor complex is diminished. In the presence of phospholipid and 5 mM Ca2+, the activities of variant and normal plasma-derived factor IX are similar; however, in the presence of activated factor VIIIa (intrinsic tenase complex), the normal augmentation of the cleavage of the specific substrate of factor IX, factor X, is not observed. The determination of the association constants for normal and variant factor IXa with factor VIIIa shows that the affinity of the activated variant factor IX for the cofactor factor VIIIa is 172-fold lower than normal. Competition studies using active site-inactivated factor IXas in the intrinsic tenase complex confirm that the defect in the variant protein is in its binding to factor VIIIa. We conclude that the structural integrity of the Gla domain of human factor IX is critical for the normal binding of factor IXa to factor VIIIa in the intrinsic tenase complex. In addition, a glycine at amino acid 12 is necessary for normal activation of factor IX by the factor VIIa-tissue factor complex

Incorporating the Cluster A and V1V2 Targets into a Minimal Structural Unit of the HIV-1 Envelope to Elicit a Cross-Clade Response with Potent Fc-Effector Functions

The generation of a potent vaccine for the prevention and/or control of HIV-1 has been unsuccessful to date, despite decades of research. Existing evidence from both infected individuals and clinical trials support a role for non-neutralizing or weakly neutralizing antibodies with potent Fc-effector functions in the prevention and control of HIV-1 infection. Vaccination strategies that induce such antibodies have proven partially successful in preventing HIV-1 infection. This is largely thought to be due to the polyclonal response that is induced in a vaccine setting, as opposed to the infusion of a single therapeutic antibody, which is capable of diverse Fc-effector functions and targets multiple but highly conserved epitopes. Here, we build on the success of our inner domain antigen, ID2, which incorporates conformational CD4-inducible (CD4i) epitopes of constant region 1 and 2 (C1C2 or Cluster A), in the absence of neutralizing antibody epitopes, into a minimal structural unit of gp120. ID2 has been shown to induce Cluster A-specific antibodies in a BALB/c mouse model with Fc-effector functions against CD4i targets. In order to generate an immunogen that incorporates both epitope targets implicated in the protective Fc-effector functions of antibodies from the only partially successful human vaccine trial, RV144, we incorporated the V1V2 domain into our ID2 antigen generating ID2-V1V2, which we used to immunize in combination with ID2. Immunized BALB/c mice generated both Cluster A- and V1V2-specific antibodies, which synergized to significantly improve the Fc-mediated effector functions compared to mice immunized with ID2 alone. The sera were able to mediate both antibody-dependent cellular cytotoxicity (ADCC) and antibody-dependent cellular phagocytosis (ADCP). We therefore conclude that ID2-V1V2 + ID2 represents a promising vaccine immunogen candidate for the induction of antibodies with optimal Fc-mediated effector functions against HIV-1

Replication-Competent NYVAC-KC Yields Improved Immunogenicity to HIV-1 Antigens in Rhesus Macaques Compared to Nonreplicating NYVAC

© 2019 Kibler et al.As part of the continuing effort to develop an effective HIV vaccine, we generated a poxviral vaccine vector (previously described) designed to improve on the results of the RV144 phase III clinical trial. The construct, NYVAC-KC, is a replication-competent, attenuated recombinant of the vaccinia virus strain NYVAC. NYVAC is a vector that has been used in many previous clinical studies but is replication deficient. Here, we report a side-by-side comparison of replication-restricted NYVAC and replication-competent NYVAC-KC in a nonhuman primate study, which utilized a prime-boost regimen similar to that of RV144. NYVAC-C and NYVAC-C-KC express the HIV-1 antigens gp140, and Gag/Gag-Pol-Nef-derived virus-like particles (VLPs) from clade C and were used as the prime, with recombinant virus plus envelope protein used as the boost. In nearly every T and B cell immune assay against HIV-1, including neutralization and antibody binding, NYVAC-C-KC induced a greater immune response than NYVAC-C, indicating that replication competence in a poxvirus may improve upon the modestly successful regimen used in the RV144 clinical trial.IMPORTANCE Though the RV144 phase III clinical trial showed promise that an effective vaccine against HIV-1 is possible, a successful vaccine will require improvement over the vaccine candidate (ALVAC) used in the RV144 study. With that goal in mind, we have tested in nonhuman primates an attenuated but replication-competent vector, NYVAC-KC, in direct comparison to its parental vector, NYVAC, which is replication restricted in human cells, similar to the ALVAC vector used in RV144. We have utilized a prime-boost regimen for administration of the vaccine candidate that is similar to the one used in the RV144 study. The results of this study indicate that a replication-competent poxvirus vector may improve upon the effectiveness of the RV144 clinical trial vaccine candidate.This investigation was funded by the Bill & Melinda Gates Foundation Poxvirus T Cell Vaccine Discovery Consortium (PTVDC) (38599). The Vaccine Immune Monitoring Centers (OPP1032144 and OPP1032325) and the Vaccine Immunology Statistical Center (OPP1032317), as part of the Collaboration for AIDS Vaccine Discovery (CAVD), were funded by the Bill & Melinda Gates Foundation. Novartis Vaccines received support for this work under contract number HHSN266200500007C from DAIDS-NIAID-NIH

Correction for Kibler et al., "Replication-Competent NYVAC-KC Yields Improved Immunogenicity to HIV-1 Antigens in Rhesus Macaques Compared to Nonreplicating NYVAC".

Volume 93, no. 3, e01513-18, 2019, https://doi.org/10.1128/JVI.01513-18. The article was originally published on 17 January 2019 with a standard copyright line (“© 2019 American Society for Microbiology. All Rights Reserved.”). We elected to pay for open access for the article after publication, necessitating replacement of the original copyright line with “© 2019 Kibler et al. This is an open-access article distributed under the terms of the Creative Commons Attribution 4.0 International license.” This change was made to the online version of the article on 21 August 2019

Replication-Competent NYVAC-KC Yields Improved Immunogenicity to HIV-1 Antigens in Rhesus Macaques Compared to Nonreplicating NYVAC

As part of the continuing effort to develop an effective HIV vaccine, we generated a poxviral vaccine vector (previously described) designed to improve on the results of the RV144 phase III clinical trial. The construct, NYVAC-KC, is a replication-competent, attenuated recombinant of the vaccinia virus strain NYVAC. NYVAC is a vector that has been used in many previous clinical studies but is replication deficient. Here, we report a side-by-side comparison of replication-restricted NYVAC and replication-competent NYVAC-KC in a nonhuman primate study, which utilized a prime-boost regimen similar to that of RV144. NYVAC-C and NYVAC-C-KC express the HIV-1 antigens gp140, and Gag/Gag-Pol-Nef-derived virus-like particles (VLPs) from clade C and were used as the prime, with recombinant virus plus envelope protein used as the boost. In nearly every T and B cell immune assay against HIV-1, including neutralization and antibody binding, NYVAC-C-KC induced a greater immune response than NYVAC-C, indicating that replication competence in a poxvirus may improve upon the modestly successful regimen used in the RV144 clinical trial. IMPORTANCE Though the RV144 phase III clinical trial showed promise that an effective vaccine against HIV-1 is possible, a successful vaccine will require improvement over the vaccine candidate (ALVAC) used in the RV144 study. With that goal in mind, we have tested in nonhuman primates an attenuated but replication-competent vector, NYVAC-KC, in direct comparison to its parental vector, NYVAC, which is replication restricted in human cells, similar to the ALVAC vector used in RV144. We have utilized a prime-boost regimen for administration of the vaccine candidate that is similar to the one used in the RV144 study. The results of this study indicate that a replication-competent poxvirus vector may improve upon the effectiveness of the RV144 clinical trial vaccine candidate

Priming with a Potent HIV-1 DNA Vaccine Frames the Quality of Immune Responses prior to a Poxvirus and Protein Boost

The use of heterologous immunization regimens and improved vector systems has led to increases in immunogenicity of HIV-1 vaccine candidates in nonhuman primates. In order to resolve interrelations between different delivery modalities, three different poxvirus boost regimens were compared. Three groups of rhesus macaques were each primed with the same DNA vaccine encoding Gag, Pol, Nef, and gp140. The groups were then boosted with either the vaccinia virus strain NYVAC or a variant with improved replication competence in human cells, termed NYVAC-KC. The latter was administered either by scarification or intramuscularly. Finally, macaques were boosted with adjuvanted gp120 protein to enhance humoral responses. The regimen elicited very potent CD4+ and CD8+ T cell responses in a well-balanced manner, peaking 2 weeks after the boost. T cells were broadly reactive and polyfunctional. All animals exhibited antigen-specific humoral responses already after the poxvirus boost, which further increased following protein administration. Polyclonal reactivity of IgG antibodies was highest against HIV-1 clade C Env proteins, with considerable cross-reactivity to other clades. Substantial effector functional activities (antibody-dependent cell-mediated cytotoxicity and antibody-dependent cell-mediated virus inhibition) were observed in serum obtained after the last protein boost. Notably, major differences between the groups were absent, indicating that the potent priming induced by the DNA vaccine initially framed the immune responses in such a way that the subsequent boosts with NYVAC and protein led only to an increase in the response magnitudes without skewing the quality. This study highlights the importance of selecting the best combination of vector systems in heterologous prime-boost vaccination regimens.This investigation was funded by the Bill & Melinda Gates Foundation Poxvirus T-Cell Vaccine Discovery Consortium (PTVDC) (38599). The Vaccine Immune Monitoring Centers (OPP1032144 and OPP1032325) and the Vaccine Immunology Statistical Center (OPP1032317), as part of the Collaboration for AIDS Vaccine Discovery (CAVD), were funded by the Bill & Melinda Gates Foundation. Novartis Vaccines received support for this work under contract number HHSN266200500007C from DAIDS-NIAID-NIH

Priming with a Potent HIV-1 DNA Vaccine Frames the Quality of Immune Responses prior to a Poxvirus and Protein Boost

The use of heterologous immunization regimens and improved vector systems has led to increases in immunogenicity of HIV-1 vaccine candidates in non-human primates. In order to resolve interrelations between different delivery modalities, three different poxvirus boost regimens were compared. Three groups of rhesus macaques were each primed with the same DNA vaccine encoding Gag, Pol, Nef, and gp140. The groups were then boosted with either the vaccinia virus strain NYVAC or a variant with improved replication competence in human cells, termed NYVAC-KC. The latter was administered either by scarification or intra-muscularly. Finally, macaques were boosted with adjuvanted gp120 protein to enhance humoral responses. The regimen elicited very potent CD4(+) and CD8(+) T cell responses in a well-balanced manner, peaking 2 weeks after the boost. T cells were broadly reactive and polyfunctional. All animals exhibited antigen-specific humoral responses already after the poxvirus boost, which further increased following protein administration. Polyclonal reactivity of IgG antibodies was highest against HIV-1 clade C Env proteins, with considerable cross-reactivity to other clades. Substantial effector functional activities (antibody-dependent cell-mediated cytotoxicity and antibody-dependent cell-mediated virus inhibition) were observed in serum obtained after the last protein boost. Notably, major differences between the groups were absent, indicating that the potent priming induced by the DNA vaccine initially framed the immune responses in such a way that the subsequent boosts with NYVAC and protein led only to an increase in the response magnitudes without skewing the quality. This study highlights the importance of selecting the best combination of vector systems in heterologous prime-boost vaccination regimens. IMPORTANCE The evaluation of HIV vaccine efficacy trials indicates that protection would most likely correlate with a polyfunctional immune response involving several effector functions from all arms of the immune system. Heterologous prime-boost regimens have been shown to elicit vigorous T cell and antibody responses in non-human primates that, however, qualitatively and quantitatively differ depending on the respective vector systems used. The present study evaluated a DNA prime and poxvirus and protein boost regimen and compared how two poxvirus vectors with various degrees of replication capacity and two different delivery modalities-conventional intramuscular delivery and percutaneous delivery by scarification-impact several immune effectors. It was found that despite the different poxvirus boosts, the overall immune responses in the three groups were similar, suggesting the potent DNA priming as the major determining factor of immune responses. These findings emphasize the importance of selecting optimal priming agents in heterologous prime-boost vaccination settings

Viral vector delivered immunogen focuses HIV-1 antibody specificity and increases durability of the circulating antibody recall response.

The modestly efficacious HIV-1 vaccine regimen (RV144) conferred 31% vaccine efficacy at 3 years following the four-shot immunization series, coupled with rapid waning of putative immune correlates of decreased infection risk. New strategies to increase magnitude and durability of protective immunity are critically needed. The RV305 HIV-1 clinical trial evaluated the immunological impact of a follow-up boost of HIV-1-uninfected RV144 recipients after 6-8 years with RV144 immunogens (ALVAC-HIV alone, AIDSVAX B/E gp120 alone, or ALVAC-HIV + AIDSVAX B/E gp120). Previous reports demonstrated that this regimen elicited higher binding, antibody Fc function, and cellular responses than the primary RV144 regimen. However, the impact of the canarypox viral vector in driving antibody specificity, breadth, durability and function is unknown. We performed a follow-up analysis of humoral responses elicited in RV305 to determine the impact of the different booster immunogens on HIV-1 epitope specificity, antibody subclass, isotype, and Fc effector functions. Importantly, we observed that the ALVAC vaccine component directly contributed to improved breadth, function, and durability of vaccine-elicited antibody responses. Extended boosts in RV305 increased circulating antibody concentration and coverage of heterologous HIV-1 strains by V1V2-specific antibodies above estimated protective levels observed in RV144. Antibody Fc effector functions, specifically antibody-dependent cellular cytotoxicity and phagocytosis, were boosted to higher levels than was achieved in RV144. V1V2 Env IgG3, a correlate of lower HIV-1 risk, was not increased; plasma Env IgA (specifically IgA1), a correlate of increased HIV-1 risk, was elevated. The quality of the circulating polyclonal antibody response changed with each booster immunization. Remarkably, the ALVAC-HIV booster immunogen induced antibody responses post-second boost, indicating that the viral vector immunogen can be utilized to selectively enhance immune correlates of decreased HIV-1 risk. These results reveal a complex dynamic of HIV-1 immunity post-vaccination that may require careful balancing to achieve protective immunity in the vaccinated population. Trial registration: RV305 clinical trial (ClinicalTrials.gov number, NCT01435135). ClinicalTrials.gov Identifier: NCT00223080