Contributions of the gp120 Variable Loops to Envelope Glycoprotein Trimer Stability in Primate Lentiviruses

Human Immunodeficiency Virus (HIV) is the etiological agent of AIDS and is responsible for the AIDS pandemic worldwide. According to UNAIDS, as of 2016, there were approximately 36.7 million people living with HIV globally, and 1.8 million new infections that year. While antiretroviral therapies and education continue to reduce these numbers, a preventative vaccine is still required to curb this epidemic. The envelope glycoprotein trimer of HIV, which is the sole protein on the surface of the virus and facilitates entry of the virus into host cells, is of keen interest to the HIV vaccine and drug-development field. Recently, the structure of this trimer was solved which has opened the door for the design of new immunogens and drugs. These solved envelope protein trimer structures demonstrate that the major variable loops of the env protein are positioned at the apex of the trimer where they play a role in trimer stability. Data from our lab has shown previously that both the V2 and V3 loop contain elements that are critical for envelope trimer stability. The work presented in this dissertation expands on these initial studies and confirms that the V2 and V3 loops contribute to envelope trimer stability and functionality in HIV-1, HIV-2 and in Simian Immunodeficiency Virus (SIV) species. We have demonstrated that the hydrophobic patch near the tip of the V3 loop, which is present in all primate lentiviruses, is critical for trimer stability in subtype C HIV-1, HIV-2 and in SIV species. Additionally, we further explored the role of the twin-cysteine motif found in the V2 loop of HIV-2 and SIV species, but not in HIV-1. We have shown that this motif is critical for the stability of the envelope protein of these viruses. Both observations are supported by our experimental data and by molecular modeling. This work supports the released trimer structures and provides a further understanding of the forces that stabilize the envelope trimer of primate lentiviruses, and will aid in the development of stabilized envelope trimer based immunogens in the effort to develop an effective HIV vaccine. Advisor: Shi-Hua Xian

\u3ci\u3eEscherichia coli\u3c/i\u3e Surface Display of Single-Chain Antibody VRC01 against HIV-1 infection

Human immunodeficiency virus type 1 (HIV-1) transmission and infection occur mainly via the mucosal surfaces. The commensal bacteria residing in these surfaces can potentially be employed as a vehicle for delivering inhibitors to prevent HIV-1 infection. In this study, we have employed a bacteria-based strategy to display a broadly neutralizing antibody VRC01, which could potentially be used to prevent HIV-1 infection. The VRC01 antibody mimics CD4-binding to gp120 and has broadly neutralization activities against HIV-1. We have designed a construct that can express the fusion peptide of the scFv-VRC01 antibody together with the autotransporter β-barrel domain of IgAP gene from Neisseria gonorrhoeae, which enabled surface display of the antibody molecule. Our results indicate that the scFv-VRC01 antibody molecule was displayed on the surface of the bacteria as demonstrated by flow cytometry and immunofluorescence microscopy. The engineered bacteria can capture HIV-1 particles via surface-binding and inhibit HIV-1 infection in cell culture

Contribution of the gp120 V3 loop to envelope glycoprotein trimer stability in primate immunodeficiency viruses

The V3 loop of the human immunodeficiency virus type 1 (HIV-1) gp120 exterior envelope glycoprotein (Env) becomes exposed after CD4 binding and contacts the coreceptor to mediate viral entry. Prior to CD4 engagement, a hydrophobic patch located at the tip of the V3 loop stabilizes the non-covalent association of gp120 with the Env trimer of HIV-1 subtype B strains. Here, we show that this conserved hydrophobic patch (amino acid residues 307, 309 and 317) contributes to gp120-trimer association in HIV-1 subtype C, HIV-2 and SIV. Changes that reduced the hydrophobicity of these V3 residues resulted in increased gp120 shedding and decreased Envmediated cell-cell fusion and virus entry in the different primate immunodeficiency viruses tested. Thus, the hydrophobic patch is an evolutionarily conserved element in the tip of the gp120 V3 loop that plays an essential role in maintaining the stability of the pre-triggered Env trimer in diverse primate immunodeficiency viruses

Models of classroom assessment for course-based research experiences

Course-based research pedagogy involves positioning students as contributors to authentic research projects as part of an engaging educational experience that promotes their learning and persistence in science. To develop a model for assessing and grading students engaged in this type of learning experience, the assessment aims and practices of a community of experienced course-based research instructors were collected and analyzed. This approach defines four aims of course-based research assessment—(1) Assessing Laboratory Work and Scientific Thinking; (2) Evaluating Mastery of Concepts, Quantitative Thinking and Skills; (3) Appraising Forms of Scientific Communication; and (4) Metacognition of Learning—along with a set of practices for each aim. These aims and practices of assessment were then integrated with previously developed models of course-based research instruction to reveal an assessment program in which instructors provide extensive feedback to support productive student engagement in research while grading those aspects of research that are necessary for the student to succeed. Assessment conducted in this way delicately balances the need to facilitate students’ ongoing research with the requirement of a final grade without undercutting the important aims of a CRE education

Contributions of the gp120 Variable Loops to Envelope Glycoprotein Trimer Stability in Primate Lentiviruses

Human Immunodeficiency Virus (HIV) is the etiological agent of AIDS and is responsible for the AIDS pandemic worldwide. According to UNAIDS, as of 2016, there were approximately 36.7 million people living with HIV globally, and 1.8 million new infections that year. While antiretroviral therapies and education continue to reduce these numbers, a preventative vaccine is still required to curb this epidemic. The envelope glycoprotein trimer of HIV, which is the sole protein on the surface of the virus and facilitates entry of the virus into host cells, is of keen interest to the HIV vaccine and drug-development field. Recently, the structure of this trimer was solved which has opened the door for the design of new immunogens and drugs. These solved envelope protein trimer structures demonstrate that the major variable loops of the env protein are positioned at the apex of the trimer where they play a role in trimer stability. Data from our lab has shown previously that both the V2 and V3 loop contain elements that are critical for envelope trimer stability. The work presented in this dissertation expands on these initial studies and confirms that the V2 and V3 loops contribute to envelope trimer stability and functionality in HIV-1, HIV-2 and in Simian Immunodeficiency Virus (SIV) species. We have demonstrated that the hydrophobic patch near the tip of the V3 loop, which is present in all primate lentiviruses, is critical for trimer stability in subtype C HIV-1, HIV-2 and in SIV species. Additionally, we further explored the role of the twin-cysteine motif found in the V2 loop of HIV-2 and SIV species, but not in HIV-1. We have shown that this motif is critical for the stability of the envelope protein of these viruses. Both observations are supported by our experimental data and by molecular modeling. This work supports the released trimer structures and provides a further understanding of the forces that stabilize the envelope trimer of primate lentiviruses, and will aid in the development of stabilized envelope trimer based immunogens in the effort to develop an effective HIV vaccine. Advisor: Shi-Hua Xian

Contributions of the gp120 Variable Loops to Envelope Glycoprotein Trimer Stability in Primate Lentiviruses

Human Immunodeficiency Virus (HIV) is the etiological agent of AIDS and is responsible for the AIDS pandemic worldwide. According to UNAIDS, as of 2016, there were approximately 36.7 million people living with HIV globally, and 1.8 million new infections that year. While antiretroviral therapies and education continue to reduce these numbers, a preventative vaccine is still required to curb this epidemic. The envelope glycoprotein trimer of HIV, which is the sole protein on the surface of the virus and facilitates entry of the virus into host cells, is of keen interest to the HIV vaccine and drug-development field. Recently, the structure of this trimer was solved which has opened the door for the design of new immunogens and drugs. These solved envelope protein trimer structures demonstrate that the major variable loops of the env protein are positioned at the apex of the trimer where they play a role in trimer stability. Data from our lab has shown previously that both the V2 and V3 loop contain elements that are critical for envelope trimer stability. The work presented in this dissertation expands on these initial studies and confirms that the V2 and V3 loops contribute to envelope trimer stability and functionality in HIV-1, HIV-2 and in Simian Immunodeficiency Virus (SIV) species. We have demonstrated that the hydrophobic patch near the tip of the V3 loop, which is present in all primate lentiviruses, is critical for trimer stability in subtype C HIV-1, HIV-2 and in SIV species. Additionally, we further explored the role of the twin-cysteine motif found in the V2 loop of HIV-2 and SIV species, but not in HIV-1. We have shown that this motif is critical for the stability of the envelope protein of these viruses. Both observations are supported by our experimental data and by molecular modeling. This work supports the released trimer structures and provides a further understanding of the forces that stabilize the envelope trimer of primate lentiviruses, and will aid in the development of stabilized envelope trimer based immunogens in the effort to develop an effective HIV vaccine

Contributions of the gp120 Variable Loops to Envelope Glycoprotein Trimer Stability in Primate Lentiviruses

Human Immunodeficiency Virus (HIV) is the etiological agent of AIDS and is responsible for the AIDS pandemic worldwide. According to UNAIDS, as of 2016, there were approximately 36.7 million people living with HIV globally, and 1.8 million new infections that year. While antiretroviral therapies and education continue to reduce these numbers, a preventative vaccine is still required to curb this epidemic. The envelope glycoprotein trimer of HIV, which is the sole protein on the surface of the virus and facilitates entry of the virus into host cells, is of keen interest to the HIV vaccine and drug-development field. Recently, the structure of this trimer was solved which has opened the door for the design of new immunogens and drugs. These solved envelope protein trimer structures demonstrate that the major variable loops of the env protein are positioned at the apex of the trimer where they play a role in trimer stability. Data from our lab has shown previously that both the V2 and V3 loop contain elements that are critical for envelope trimer stability. The work presented in this dissertation expands on these initial studies and confirms that the V2 and V3 loops contribute to envelope trimer stability and functionality in HIV-1, HIV-2 and in Simian Immunodeficiency Virus (SIV) species. We have demonstrated that the hydrophobic patch near the tip of the V3 loop, which is present in all primate lentiviruses, is critical for trimer stability in subtype C HIV-1, HIV-2 and in SIV species. Additionally, we further explored the role of the twin-cysteine motif found in the V2 loop of HIV-2 and SIV species, but not in HIV-1. We have shown that this motif is critical for the stability of the envelope protein of these viruses. Both observations are supported by our experimental data and by molecular modeling. This work supports the released trimer structures and provides a further understanding of the forces that stabilize the envelope trimer of primate lentiviruses, and will aid in the development of stabilized envelope trimer based immunogens in the effort to develop an effective HIV vaccine

Characterization of a Dual-tropic Human Immunodeficiency Virus (HIV-1) Strain Derived from the Prototypical X4 Isolate HXBc2

Human immunodeficiency virus type 1 (HIV-1) coreceptor usage and tropism can be modulated by the V3 loop sequence of the gp120 exterior envelope glycoprotein. For coreceptors, R5 viruses use CCR5, X4 viruses use CXCR4, and dual-tropic (R5X4) viruses use either CCR5 or CXCR4. To understand the requirements for dual tropism, we derived and analyzed a dual-tropic variant of an X4 virus. Changes in the V3 base, which allow gp120 to interact with the tyrosine-sulfated CCR5 N-terminus, and deletion of residues 310/311 in the V3 tip were necessary for efficient CCR5 binding and utilization. Thus, both sets of V3 changes allowed CCR5 utilization with retention of the ability to use CXCR4. We also found that the stable association of gp120 with the trimeric envelope glycoprotein complex in R5X4 viruses, as in X4 viruses, is less sensitive to V3 loop changes than gp120-trimer association in R5 viruses

Tryptophan 375 stabilizes the outer-domain core of gp120 for HIV vaccine immunogen design

The outer-domain core of gp120 may serve as a better HIV vaccine immunogen than the full-length gp120 because of its greater stability and immunogenicity. In our previous report, we introduced two disulfide bonds to the outer-domain core of gp120 to fix its conformation into a CD4-bound state, which resulted in a significant increase in its immunogenicity when compared to the wild-type outer-domain core. In this report, to further improve the immunogenicity of the outer-domain core based immunogen, we have introduced a Tryptophan residue at gp120 amino acid sequence position 375 (375S/W). Our data from immunized guinea pigs indeed shows a striking increase in the immune response due to this stabilized core outer-domain. Therefore, we conclude that the addition of 375W to the outer-domain core of gp120 further stabilizes the structure of immunogen and increases the immunogenicit

\u3ci\u3eEscherichia coli\u3c/i\u3e Surface Display of Single-Chain Antibody VRC01 against HIV-1 infection

Human immunodeficiency virus type 1 (HIV-1) transmission and infection occur mainly via the mucosal surfaces. The commensal bacteria residing in these surfaces can potentially be employed as a vehicle for delivering inhibitors to prevent HIV-1 infection. In this study, we have employed a bacteria-based strategy to display a broadly neutralizing antibody VRC01, which could potentially be used to prevent HIV-1 infection. The VRC01 antibody mimics CD4-binding to gp120 and has broadly neutralization activities against HIV-1. We have designed a construct that can express the fusion peptide of the scFv-VRC01 antibody together with the autotransporter β-barrel domain of IgAP gene from Neisseria gonorrhoeae, which enabled surface display of the antibody molecule. Our results indicate that the scFv-VRC01 antibody molecule was displayed on the surface of the bacteria as demonstrated by flow cytometry and immunofluorescence microscopy. The engineered bacteria can capture HIV-1 particles via surface-binding and inhibit HIV-1 infection in cell culture