Progress 06/01/10 to 05/31/15
Outputs
Target Audience:
Nothing Reported
Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?This project was the thesis of one PhD student, For Yue Tso who is now a postdoctoral fellow. How have the results been disseminated to communities of interest?The results were presented at scientific meeting, and some of the findings were published previously. What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
The objective of this project remained unchanged for the past several years; it is to determine whether infection of non-human primate by SHIV, a chimeric virus between the simian immunodeficiency virus (SIV) and the human immunodeficiency virus (HIV) can be used as a model to study lentiviruses evolution during the disease course. Our study has been making use of an ongoing research project to study perinatal transmission of HIV-1 and other viruses in Zambia. The specific aims are: 1) To characterize changes in the envelope (Env) proteins of clade C HIV-1 isolated from infants at different time points in the disease course, and correlate these changes to disease progression. 2) To characterize the Env proteins of SHIV isolated longitudinally over the disease course in infected animals and correlate these changes to disease progression. Aim one has been completed in the past several years and we have been focusing on the second aim in the past two years, and the project is now essentially been completed. For the past year we have focused on completing aim 2, which is to determine the biological variations that occurred to the clade C Env during disease progression. We hypothesized that HIV envelope variants from different stages of the disease might have different biological properties. Our macaque model allows studying the longitudinal changes of the biological functions of the V1-V5 region of Env from an infectious molecular clone inoculum over the entire disease course. Our hypothesis is that envelopes from various disease stages have different biological characteristics, which might confer fitness advantages for the virus. We have focused on an animal RPn-8 which was infected by the SHIV. As shown previously, RPn-8 maintained a high viral load (>1x104 copies/ml) throughout the disease course (42) and the CD4+ T-cell counts gradually declined to <200 cells/ul from ~28 months post infection (mpi) onwards. Our analysis showed a significant increase in the net charge of V1-V5 region during early infection (6 mpi) compared to the inoculum However, the net charge decreased substantially by 37 mpi when the CD4+ T-cell counts fell to <200 cells/ul. Surprisingly, as RPn-8 had advanced AIDS, the net charge of the V1-V5 region recovered to pre-AIDS levels. We then determine the susceptibility of the SHIV Env V1-V5 region to neutralizing antibodies using contemporaneous and non-contemporaneous plasma from RPn-8. We found that neither the naïve nor the contemporaneous plasma could neutralize any of the envelopes tested. However, non-contemporaneous plasma was able to neutralize envelopes from earlier time points, but this capability tended to decrease with disease progression. This was further confirmed by the examination of 6 mpi envelopes against plasma samples from all later time points, where the 64 mpi plasma had the weakest neutralizing ability. In addition, our data show that envelopes from 6 mpi were mostly sensitive to broadly neutralizing monoclonal antibody. However, the majority of the envelopes displayed increased mAb resistance by 37 mpi. Surprisingly, mAb-sensitive envelopes re-emerged at 64 mpi. To determine if envelopes derived from different disease stages could affect the viral replicative fitness and its relationship with neutralization sensitivity, we examined the replication kinetics of viruses expressing various envelope clones in peripheral blood mononuclear cells. Viruses with 6 month envelopes displayed distinctively faster ex-vivo replication (P = 0.0008) than those with 64 mpi envelopes. In contrast, viruses bearing envelopes from later time points showed slightly slower ex-vivo replication compared to the inoculum over time. A possible factor that can influence ex-vivo replication is divergence in the envelope-CD4 binding capacity. To this end, our in-vitro CD4 binding assay showed that the CD4 binding proficiency of envelope improved with disease progression. Surprisingly, this enhanced CD4 binding capability was not maintained and reverted to pre-AIDS levels during advanced AIDS. Finally, we determined the fusiongenicity of the viral Env from different time points, since fusion of viral envelope to the target cell is a critical rate-limiting step that could affect ex-vivo replication. A FRET-based virus to cell fusion assay was used to examine the fusion efficiency of envelopes from various time points. Our result showed that viruses with 6 mpi envelopes, had significantly enhanced fusion abilities compared with the inoculum (P = < 0.0001). Envelopes isolated from 37 mpi displayed a diverse fusion capability. Additionally, this weak fusion phenotype dominated among envelopes isolates from advanced AIDS. This suggests that envelope fusion ability increased during early infection, but gradually declined with disease progression.
Publications
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Progress 10/01/13 to 09/30/14
Outputs
Target Audience:
Nothing Reported
Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided? This project was the thesis of one PhD student, For Yue Tso who is now a postdoctoral fellow. How have the results been disseminated to communities of interest? The results were presented at scientific meeting, and some of the findings were published previously. What do you plan to do during the next reporting period to accomplish the goals? Our plan is to complete the study aim 2 inorder to wrap up the project.
Impacts
What was accomplished under these goals?
The objective of this project remained unchanged for the past several years; it is to determine whether infection of non-human primate by SHIV, a chimeric virus between the simian immunodeficiency virus (SIV) and the human immunodeficiency virus (HIV) can be used as a model to study lentiviruses evolution during the disease course. Our study has been making use of an ongoing research project to study perinatal transmission of HIV-1 and other viruses in Zambia. The specific aims are: 1) To characterize changes in the envelope (Env) proteins of clade C HIV-1 isolated from infants at different time points in the disease course, and correlate these changes to disease progression. 2) To characterize the Env proteins of SHIV isolated longitudinally over the disease course in infected animals and correlate these changes to disease progression. Aim one has been completed in the past several years and we have been focusing on the second aim in the past two years, and the project is now essentially been completed. For the past year we have focused on completing aim 2, which is to determine the biological variations that occurred to the clade C Env during disease progression. We hypothesized that HIV envelope variants from different stages of the disease might have different biological properties. Our macaque model allows studying the longitudinal changes of the biological functions of the V1-V5 region of Env from an infectious molecular clone inoculum over the entire disease course. Our hypothesis is that envelopes from various disease stages have different biological characteristics, which might confer fitness advantages for the virus. We have focused on an animal RPn-8 which was infected by the SHIV. As shown previously, RPn-8 maintained a high viral load (>1x104 copies/ml) throughout the disease course (42) and the CD4+ T-cell counts gradually declined to <200 cells/ul from ~28 months post infection (mpi) onwards. Our analysis showed a significant increase in the net charge of V1-V5 region during early infection (6 mpi) compared to the inoculum However, the net charge decreased substantially by 37 mpi when the CD4+ T-cell counts fell to <200 cells/ul. Surprisingly, as RPn-8 had advanced AIDS, the net charge of the V1-V5 region recovered to pre-AIDS levels. We then determine the susceptibility of the SHIV Env V1-V5 region to neutralizing antibodies using contemporaneous and non-contemporaneous plasma from RPn-8. We found that neither the naïve nor the contemporaneous plasma could neutralize any of the envelopes tested. However, non-contemporaneous plasma was able to neutralize envelopes from earlier time points, but this capability tended to decrease with disease progression. This was further confirmed by the examination of 6 mpi envelopes against plasma samples from all later time points, where the 64 mpi plasma had the weakest neutralizing ability. In addition, our data show that envelopes from 6 mpi were mostly sensitive to broadly neutralizing monoclonal antibody. However, the majority of the envelopes displayed increased mAb resistance by 37 mpi. Surprisingly, mAb-sensitive envelopes re-emerged at 64 mpi. To determine if envelopes derived from different disease stages could affect the viral replicative fitness and its relationship with neutralization sensitivity, we examined the replication kinetics of viruses expressing various envelope clones in peripheral blood mononuclear cells. Viruses with 6 month envelopes displayed distinctively faster ex-vivo replication (P = 0.0008) than those with 64 mpi envelopes. In contrast, viruses bearing envelopes from later time points showed slightly slower ex-vivo replication compared to the inoculum over time. A possible factor that can influence ex-vivo replication is divergence in the envelope-CD4 binding capacity. To this end, our in-vitro CD4 binding assay showed that the CD4 binding proficiency of envelope improved with disease progression. Surprisingly, this enhanced CD4 binding capability was not maintained and reverted to pre-AIDS levels during advanced AIDS. Finally, we determined the fusiongenicity of the viral Env from different time points, since fusion of viral envelope to the target cell is a critical rate-limiting step that could affect ex-vivo replication. A FRET-based virus to cell fusion assay was used to examine the fusion efficiency of envelopes from various time points. Our result showed that viruses with 6 mpi envelopes, had significantly enhanced fusion abilities compared with the inoculum (P = < 0.0001). Envelopes isolated from 37 mpi displayed a diverse fusion capability. Additionally, this weak fusion phenotype dominated among envelopes isolates from advanced AIDS. This suggests that envelope fusion ability increased during early infection, but gradually declined with disease progression.
Publications
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Progress 10/01/12 to 09/30/13
Outputs
Target Audience:
Nothing Reported
Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?
Nothing Reported
How have the results been disseminated to communities of interest? Through reports at professional meetings and by through peer-reviewed publications of our findings. What do you plan to do during the next reporting period to accomplish the goals? Will continue to explore the role of viral Env evoluction during disease progression using animal models, and explore the pssibility of using anti-viral drug to cure infections.
Impacts
What was accomplished under these goals?
This past year we have focused on aim 2 which is to determine the biological variations that occurred to the clade C Env during disease progression. We hypothesized that HIV envelope variants from different stages of the disease might have different biological properties. We have previously shown a strikingly similar molecular evolution of the HIV-1 clade C (HIV-C) envelope during infection in a Zambian infant (1157i) and an infant rhesus macaque (RPn-8) infected by SHIV-1157i, a SHIV expressing the recently transmitted HIV-C envelope of 1157i [8]. This finding validated our macaque model for studying envelope evolution during disease progression. Importantly, our macaque model allows studying the longitudinal changes of the biological functions of the V1-V5 region from an infectious molecular clone inoculum over the entire disease course. Moreover, significant mutations were observed within the HIV-C envelope sequences from different disease stages in the infected macaque RPn-8 [8]. However, whether these envelope mutations translate into different biological functions remains unanswered. Therefore, we addressed this question by characterizing the biological properties of the V1-V5 region cloned from RPn-8 during disease progression. Our hypothesis is that envelopes from various disease stages have different biological characteristics, which might confer fitness advantages for the virus. This was carried out by cloning the Env region from a SHIV infected rhesus macaque at various time points, from initial infection through disease progression and AIDS. The Env clones were tested for their neutralizing characteristics against autologous plasma and pooled monoclonal antibodies. Infection of peripheral blood mononuclear cells with provirus expressing the Env clones was used to examine the effect of various Env on the viral replication kinetics. Envelope content on the virus particles was also measured. Cell surface biotinlyation of Env transfected cells was used to evaluate Env cleavage, and CD4 binding affinity was determined by CD4 binding assay. We found that the envelopes from early infection tended to be more positively charged and neutralization sensitive, but the net charge decreased and Env became neutralization resistant with time. Early envelopes also exhibited better cleavage, higher gp120 content on virion, superior fusion capacity and faster replication kinetics compared with envelopes from later time points. Lastly, CD4 binding tended to improve as disease progressed, but reverted to early infection level after the onset of AIDS. Envelope gained distinctive biological characteristics as disease progressed. Potentially beneficiary functions could have arisen during early infection for better in-vivo viral fitness. However, some Env properties reverted to pre-AIDS levels in the absence of immune pressure.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2013
Citation:
Tso, FY., Abrahamyan, L., Hu, S H., Ruprecht, R. and Wood, C. 2013. Variations in the biological functions of HIV-1 clade C envelope in a SHIV-infected rhesus macaque during disease progression. PLoS One, 8 (6):e66973. PMCID: PMC3694120
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Progress 10/01/11 to 09/30/12
Outputs
OUTPUTS: The overall objective of this project is to determine whether infection of non-human primate by SHIV, a chimeric virus between the simian immunodeficiency virus (SIV) and the human immunodeficiency virus (HIV) and the resultant evolution of the virus during the disease course, is a suitable model for studying lentiviruses such as HIV-1 evolution in humans, such model can further be used to study evolution of lentiviruses that infect food animal, such as bovine immunodeficiency virus (BIV), the ovine progressive pneumonia virus (OPPV) of sheep, and caprine arthritis-encephalitis virus (CAEV) of goats. Our immediate goal is to longitudinally isolate and sequence clade C HIV-1 env from perinatally infected infants peripheral blood cell DNA and to characterize the patterns and within-host diversification of the gene with time, and compared that to the evolution and changes of the same gene with a SHIV in the macaque model. Our study has been making use of an ongoing research project to study perinatal transmission of HIV-1 and other viruses in Zambia. The specific aims are: 1) To characterize changes in the Env proteins of clade C HIV-1 isolated from infants at different time points in the disease course, and correlate these changes to disease progression. 2) To characterize the Env proteins of SHIV isolated longitudinally over the disease course in infected animals and correlate these changes to disease progression. This past year we have focused on aim 2 which is to determine the biological variations that occurred to the clade C Env during disease progression. We hypothesized that HIV envelope variants from different stages of the disease might have different biological properties. This was carried out by cloning the Env region from a SHIV infected rhesus macaque at various time points, from initial infection through disease progression and AIDS. The Env clones were tested for their neutralizing characteristics against autologous plasma and pooled monoclonal antibodies. Infection of peripheral blood mononuclear cells with provirus expressing the Env clones was used to examine the effect of various Env on the viral replication kinetics. Envelope content on the virus particles was also measured. Cell surface biotinlyation of Env transfected cells was used to evaluate Env cleavage, and CD4 binding affinity was determined by CD4 binding assay. Our neutralization data indicated that the Env tends to be of the sensitive phenotype early in the infection, but evolved to be more resistant over time. Infection of cells showed that virus with Env variants from early stages of infection have faster replication kinetics; biotinlyation results indicated an increase in the gp160 to gp120 ratios over time, suggesting the Env became less cleavable as disease progressed. This resulted in diminished incorporation of envelope proteins into HIV virus particles over time. However, the decrease did not affect the CD4 binding capability of the envelopes. In fact, CD4 binding tends to improve as disease progressed, but reverted to early infection level after onset of AIDS. PARTICIPANTS: Nothing significant to report during this reporting period. TARGET AUDIENCES: Nothing significant to report during this reporting period. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
Our study showed that by using a chimeric lentivirus of human and simian to study disease progression in macaque is a relevant model for the human disease. We have shown that variations in the biological properties of the envelope occurred during disease progression. Beneficiary properties, such as faster replication kinetics and improved CD4 binding, are gained in early infection for better survival against immune selection. However, such functional gains were reversed after the animal developed AIDS, which is likely due the depletion of immune response and selection pressure. This is in addition to what we have previously shown, that viral envelope diversifications during disease progression are different between infected macaques that showed disease progression and non-progression phenotypes. The fact that non-progressor animal had envelope binding antibodies but no neutralizing antibodies against the virus indicated that neutralizing antibody responses are not the main mechanism behind its control over the virus. There may be other host factors, including the cell mediated immune response against the virus that could play a role in suppressing viral infection. Since the well-being of the infected host is a competition between the host response and the viral attempt to mutate and escape the response. Therefore, a better understanding of the viral mutations, especially those that occurred in the viral envelope glycoprotein, and their effect on envelope functions during the disease course is important. Further deciphering of these factors may lead to the development of strategies to prevent disease progression. designs for both humans and food animals such as bovine. Understanding the genetic variations in viral envelope during disease progression is also critical for vaccine development to prevent infection of animals by lentiviruses.
Publications
- Tso, F.Y., Tully, D.C., Gonzalez, S., Quince, C., Ho, O., Polacino, P., Ruprecht, R.M., Hu, S.L., Wood, C. 2012. Dynamics of Envelope Evolution in Clade C SHIV-infected Pigtailed Macaques during Disease Progression analyzed by Ultra-deep Pyrosequencing. 2012 PLoS One. 7(3):e32827
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Progress 10/01/10 to 09/30/11
Outputs
OUTPUTS: The overall objective of this project remained unchanged. It is to determine whether infection of non-human primate by SHIV, and the resultant evolution therein, is a suitable model for studying lentiviruses such as HIV-1 evolution in humans, such model can further be used to study evolution of lentiviruses that infect food animal, such as bovine immunodeficiency virus (BIV), the ovine progressive pneumonia virus (OPPV) of sheep, and caprine arthritis-encephalitis virus (CAEV) of goats. Our immediate goal is to longitudinally isolate and sequence clade C HIV-1 env from perinatally infected infants peripheral blood cell DNA and to characterize the patterns and within-host diversification of the gene with time, and compared that to the evolution and changes of the same gene with a chimeric simian immunodeficiency virus/HIV chimeric virus in the macaque model . Our study has been making use of an ongoing research project to study perinatal transmission of HIV-1 and other viruses in mother/infant pairs from Zambia. The specific aims also remained unchanged and they are: 1) To characterize changes in the Env proteins of clade C HIV-1 isolated from infants at different time points in the disease course, and correlate these changes to disease progression. 2) To characterize the Env proteins of clade C SHIV isolated longitudinally over the disease course in infected animals and correlates these changes to disease progression. A newly developed clade C SHIV, SHIV-1157ipd3N4, which was able to infect rhesus macaques, closely resembled primary HIV-1 in transmission and pathogenesis, was used to infect several pig-tailed macaques. One of the infected animals subsequently progressed to AIDS, whereas one remained a non-progressor. Our strategy is the analyzed the differences in the evolution of the virus in the progressor versus the non-progressor animal. The viral envelope evolution in the infected animals during disease progression was analyzed by a bioinformatics approach using ultra-deep pyrosequencing. Our results showed substantial envelope variations emerging in the progressor animal after the onset of AIDS. These envelope variations impacted the length of the variable loops and charges of different envelope regions. Additionally, multiple mutations were located at the CD4 and CCR5 binding sites, potentially affecting receptor binding affinity, viral fitness and they might be selected at late stages of disease. . More importantly, these envelope mutations are not random since they had repeatedly been observed in a rhesus macaque and a human infant infected by either SHIV or HIV-1, respectively, carrying the parental envelope of the infectious molecular clone SHIV-1157ipd3N4. Moreover, similar mutations were also observed from other studies on different clades of envelopes regardless of the host species. These recurring mutations in different envelopes suggest that there is a common evolutionary pattern and selection pathway for the HIV-1 envelope during disease progression. PARTICIPANTS: Nothing significant to report during this reporting period. TARGET AUDIENCES: Nothing significant to report during this reporting period. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
Our study showed that using a chimeric lentivirus of human and simian to study disease progression in macaque is a relevant model for the human disease. In addition with this study, we provided a comprehensive view of the HIV-1 clade C envelope diversifications during disease progression between progressor and non-progressor pig-tailed macaques. Despite an extremely low level of diversity and divergence from the inoculum, we were still able to detect minor envelope variants in the non-progressor macaque over time, demonstrating the value of the ultra-deep pyrosequencing technology. However, there was clearly a mechanism by which the non-progressor animal was able to keep its infection well controlled. The fact that non-progressor animal had envelope binding antibodies but no neutralizing antibodies against SHIV-1157ipd3N4, as reported in the previous study, indicated that neutralizing antibody responses are not the main mechanism behind its control over the virus. In addition, the gradual reduction of PBMC proviral load occurred in the non-progressor without any significant decreases in its CD4+ T-cell counts, suggesting that this reduction was not due to a lack of target cells. This differs from the observation in the progressor macaque, whose reduction of PBMC proviral load coincided with low CD4+ T-cell counts. Together, the data strongly suggests that cell-mediated immunity, such as cytotoxic T lymphocyte (CTL) responses, may be responsible for suppressing viral replication in non-progressor. Since mutations occur more frequently during viral replication, a diminished virus production will minimize the level of viral genetic variation, thus explaining the lack of envelope diversity and divergence in non-progressor during the course of observation. A further understanding of host factors which caused these genetic changes in the virus and the significance of these changes on the biological function of the envelope is important. Further deciphering of these factors may lead to the development of strategies to prevent disease progression, and results derived from this study could also provide beneficial insights for future of lentivirus vaccine designs for both humans and food animals such as bovine.
Publications
- No publications reported this period
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Progress 06/01/10 to 12/31/10
Outputs
OUTPUTS: The overall objective of this project is to determine whether infection of non-human primate by SHIV, and the resultant evolution therein, is a suitable model for studying lentiviruses such as HIV-1 evolution in humans, such model can further be used to study evolution of lentiviruses that infect food animal, such as BIV. Our immediate goal is to longitudinally isolate and sequence clade C HIV-1 env from perinatally infected infants peripheral blood cell DNA and to characterize the patterns and within-host diversification of the gene. Our study has been making use of an ongoing research project to study perinatal transmission of HIV-1 and other viruses in mother/infant pairs (MIPs) from Zambia. The specific aims have remained unchanged and they are: 1) To characterize changes in the Env proteins of clade C HIV-1 isolated from infants at different time points in the disease course, and correlate these changes to disease progression. 2) To characterize the Env proteins of clade C SHIV isolated longitudinally over the disease course in infected animals and correlates these changes to disease progression. Here, an infant macaque was infected with SHIV-1157i, an R5 tropic SHIV-C which expresses a primary HIV-C envelope derived from an infected human infant, and monitored over a five-year period. Longitudinal samples collected from the infected macaque and infant enable us to examine, in parallel, the sequence variation of an identical clade C V1-V5 region arising throughout the disease course in macaque and human. Phylogenetic analysis of the V1-V5 regions of SHIV and HIV-1 derived from the infected macaque and infant at different time points post-infection, respectively, revealed that the changes in the macaque closely reflected those of infant during disease progression. Similar distribution patterns of non-synonymous substitutions and hot spots were observed between macaque and human. Analysis of possible N-linked glycosylation sites revealed several variations that are common between the virus populations in the two host species. These variations correlate with decline of CD4 count in the macaque and might be linked with disease progression. Thus, SHIV-C infection of macaque is a relevant animal model for studying variation of primary HIV-C envelope during disease progression and could be used to analyze the selection pressures that are associated with those changes. PARTICIPANTS: This project represents a continuous collaboration between UNL, the University of Zambia and Harvard University. This project has also provided training opportunities for graduate and postdoctoral students at UNL. TARGET AUDIENCES: Not relevant to this project. PROJECT MODIFICATIONS: Not relevant to this project.
Impacts
Our study showed that using a chimeric lentivirus of human and simian to study disease progression in macaque is a relevant model for the human disease. In addition, even though it is difficult to make a direct comparison of the time course between human and macaque in disease progression, mutations observed in our infected macaque at the late time point could be indicators of disease progression or a deteriorating status of the host immune system, which may predict what will also occur in disease progression in humans. A further understanding of factors which caused these genetic changes and the significance of these changes on the biological function of the envelope is important. Further deciphering of these factors may lead to the development of strategies to prevent disease progression, and results derived from this study could also provide beneficial insights for future of lentivirus vaccine designs for both humans and food animals such as bovine.
Publications
- No publications reported this period
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