Faculty Bibliography

The results of the RV144 vaccine clinical trial showed a correlation between high level of anti-V1V2 antibodies (Abs) and a decreased risk of acquiring HIV-1 infection. This turned the focus of HIV vaccine design to the induction of elevated levels of anti-V2 Abs to increase vaccine efficacy. In plasma samples from HIV-1 infected Cameroonian individuals, we observed broad variations in levels of anti-V2 Abs, and 6 of the 79 plasma samples tested longitudinally displayed substantial deficiency of V2 Abs. Sequence analysis of the V2 region from plasma viruses and multivariate analyses of V2 characteristics showed a significant difference in several features between V2-deficient and V2-reactive plasma Abs. These results suggest that HIV vaccine immunogens containing a shorter V2 region with fewer glycosylation sites and higher electrostatic charges can be beneficial for induction of a higher level of anti-V2 Abs and thus contribute to HIV vaccine efficacy.

Genetic and immunologic analyses of epidemiologically-linked HIV transmission enable insights into the impact of immune responses on clinical outcomes. Human vaccine trials and animal studies of HIV-1 infection have suggested immune correlates of protection; however, their role in natural infection in terms of protection from disease progression is mostly unknown. Four HIV-1⁺ Cameroonian individuals, three of them epidemiologically-linked in a polygamous heterosexual relationship and one incidence-matched case, were studied over 15 years for heterologous and cross-neutralizing antibody responses, antibody binding, IgA/IgG levels, antibody-dependent cellular cytotoxicity (ADCC) against cells expressing wild-type or CD4-bound Env, viral evolution, Env epitopes, and host factors including HLA-I alleles. Despite viral infection with related strains, the members of the transmission cluster experienced contrasting clinical outcomes including cases of rapid progression and long-term non-progression in the absence of strongly protective HLA-I or CCR5Δ32 alleles. Slower progression and higher CD4/CD8 ratios were associated with enhanced IgG antibody binding to native Env and stronger V1V2 antibody binding responses in the presence of viruses with residue K169 in V2. ADCC against cells expressing Env in the CD4-bound conformation in combination with low Env-specific IgA/IgG ratios correlated with better clinical outcome. This data set highlights for the first time that V1V2-directed antibody responses and ADCC against cells expressing open, CD4-exposed Env, in the presence of low plasma IgA/IgG ratios, can correlate with clinical outcome in natural infection. These parameters are comparable to the major correlates of protection, identified post-hoc in the RV144 vaccine trial; thus, they may also modulate the rate of clinical progression once infected. The findings illustrate the potential of immune correlate analysis in natural infection to guide vaccine development.

Background: HIV-1 infection of four individuals and subsequent superinfection of one of these individuals with related strains enables valuable insight into the impact of natural immune responses and host factors on clinical outcome from comparably "primed" individuals.
Method(s): Four HIV-1+ individuals, epidemiologically linked by their infecting viruses, were studied longitudinally for neutralization, antibody binding, IgA/IgG quantitation and antibody-dependent cellular cytotoxicity (ADCC). Immune responses and viral signatures were analyzed for correlates of protection and clinical parameters.
Result(s): Despite viral infection with related strains, the course of HIV-1+ infection varied greatly between each individual. For example, whereas the two slow progressors exhibited the highest neutralization, ADCC towards CD4-induced Env epitopes, SOSIP and/or V1V2 antibody binding, the two progressors had the highest envelope specific IgA/IgG ratios in plasma. One of the progressors additionally experienced superinfection with a virus that was traced back genetically to one of the slow progressors (linked superinfection). Of interest, superinfection induced immune responses comparable to the linked HIV-1+ individual including Envspecific IgA/IgG ratios, neutralization and antibody binding patterns. In the combined longitudinal data set, ADCC against cells expressing untriggered Env did not correlate with clinical parameters; however both heterologous neutralization and antibody binding against two different V1V2 scaffolded antigens were significantly associated with higher CD4/CD8 ratios.
Conclusion(s): Superinfection with a genetically linked HIV-1 strain induced comparable immune responses as observed in the related singly infected individual. Our longitudinal data set of four epidemiologically linked cases underline the importance of polyfunctional immune responses, including neutralization and V1V2 antibody binding, for beneficial clinical outcomes and possibly even protection of superinfection

Background: Since the introduction of HAART in Cameroon in 2004, there has been a considerable increase in the rate of HIV resistance associated mutations (RAMs). Although previous studies have reported the effects of HIV drug resistance (HIVDR) in both drug naive and ART patients, little is known about the emergence of additional site-specific drug resistance associated mutations.
Method(s): 4475 Cameroonian HIV nucleotide sequences were downloaded from the Los Alamos HIV database covering reverse transcriptase (RT, 1212 sequences), protease (PR, 2696) and integrase (IN, 567) genes. The sequences were assigned to periods before (1996-2003) or after (2004-now) introduction of ART in Cameroon. WebLogo and AnalyzeAlign tools were used for a comparative amino acid sequence analysis of "pre-ART" and "ART" sequences to determine the emergence and quantitative change of novel mutation sites that have not yet been associated with drug resistance. Emerging mutation sites that exhibited a statistically significant change including an at least 5-fold change and a difference of at least 5% from pre-ART to ART were structurally assessed using published crystal structures.
Result(s): According to HIV-1 CRF02-AG being the most prevalent subtype and RT inhibitors being the most frequently used drugs in Cameroon, we observed the highest number of emerging pol mutations in RT of CRF02-AG sequences (10). The emergence of novel mutations was not attributed to a phylogenetic shift. The close clustering of the emerging RT mutations around the drug binding pockets suggest an impact on HIVDR.
Conclusion(s): Our ongoing screenings within a Cameroonian cohort of HIV+ individuals aim to address the clinical relevance of the emerging pol mutations. Phenotypic drug resistance testing and clinical assessments need to confirm their contribution to HIVDR

Current serological assays that are used for cross-sectional HIV incidence estimation have been shown to misclassify individuals with chronic infection. Limited information exists on the performance of cross-sectional incidence assays in Central Africa. HIV-positive individuals from Cameroon who were infected for at least one year or two years were evaluated to determine the false recent ratio of a two-assay algorithm which includes the Limiting Antigen Avidity (LAg-Avidity) assay (ODn<1.5) and HIV viral load (>1000 copies/mL). The subject-level false recent ratio was 5.3% (95% CI, 2.1-10.5) for individuals infected for >/=1 year and 3.9% (95% CI, 0.8-11.0) for individuals infected for >/= 2 years. These data suggest that the LAg-Avidity plus viral load incidence algorithm may overestimate HIV incidence rates in Central Africa.

The global intensification of ART can lead to increased rates of HIV drug resistance (HIVDR) mutations in treated, and also in ART naive patients. ART naive HIV-1 infected patients from Cameroon were subjected to a multimethod HIVDR analysis using Amplification Refractory Mutation System (ARMS)-PCR, Sanger sequencing, and longitudinal Next-Generation Sequencing (NGS), to determine their mutation profiles for K103N, Y181C, K65R, M184V, and T215F/Y. We processed 66 ART-naive HIV-1+ patients with highly diverse subtypes, underlining the predominance of CRF02_AG and the increasing rate of F2 and other recombinant forms in Cameroon. We compared three resistance testing methods for 5 major mutation sites. Using Sanger sequencing, the overall prevalence of HIVDR mutations was 7.6% (5/66) and included all studied mutations except K65R. Comparing ARMS-PCR with Sanger sequencing as a reference, we obtained a sensitivity of 100% (5/5) and a specificity of 95% (58/61), caused by three false positive calls with ARMS-PCR. For 32/66 samples we obtained NGS data and we observed two additional mismatches made up by minority variants (7%, 18%) that might not be clinically relevant. Longitudinal NGS analyses revealed changes in HIVDR mutations in all five positive subjects that could not be attributed to treatment. In one of these cases, superinfection led to the temporary masking of a resistant virus.HIVDR mutations can be sensitively detected by ARMS-PCR and Sequencing methods with comparable performance. Longitudinal changes in HIVDR mutations have to be considered even in the absence of treatment.

OBJECTIVE: To investigate the viral genetic evolution, spatial origins and patterns of transmission of HIV-1 subtype B in Shanghai, China. METHODS: A total of 242 Shanghai subtype B and 1519 reference pol sequences were subjected to phylogenetic inference and genetic transmission network analyses. RESULTS: Phylogenetic analysis revealed that subtype B strains circulating in Shanghai were genetically diverse and closely associated with viral sequence lineages in Beijing (76 of 242 [31.4%]), Central China (Henan/Hebei/Hunan/Hubei) (43 of 242 [17.8%]), Chinese Taiwan (20 of 242 [8.3%]), Japan (6 of 242 [2.5%]), and Korea (7 of 242 [2.9%]), suggesting multiple introductions into Shanghai from mainland China and Taiwan, Japan, and Korea. Interestingly, a monophyletic Shanghai lineage (SH-L) (36 of 242 [14.9%]) of HIV-1 subtype B most likely originated from an Argentine strain, transferred through Liaoning infected individuals. In-depth analyses of 195 Shanghai subtype B sequences revealed that a total of 37.9% (n = 74) sequences contributed to 35 transmission networks, whereof 33.8% (n = 25) of the sequences associated with infected individuals from other provinces. CONCLUSIONS: Our new findings reflect the evolution complexity and transmission dynamics of HIV-1 subtype B in Shanghai, which would provide critical information for the design of effective prevention measures against HIV transmission.

HIV superinfection describes the sequential infection of an individual with two or more unrelated HIV strains. Intersubtype superinfection has been shown to cause a broader and more potent heterologous neutralizing antibody response when compared to singly infected controls, yet the effects of intrasubtype superinfection remain controversial. Longitudinal samples were analyzed phylogenetically for pol and env regions using Next-Generation Sequencing and envelope cloning. The impact of CRF02_AG intrasubtype superinfection was assessed for heterologous neutralization and antibody binding responses. We compared two cases of CRF02_AG intrasubtype superinfection that revealed complete replacement of the initial virus by superinfecting CRF02_AG variants with signs of recombination. NYU6564, who became superinfected at an early time point, exhibited greater changes in antibody binding profiles and generated a more potent neutralizing antibody response post-superinfection compared to NYU6501. In contrast, superinfection occurred at a later time point in NYU6501 with strains harboring significantly longer V1V2 regions with no observable changes in neutralization patterns. Here we show that CRF02_AG intrasubtype superinfection can induce a cross-subtype neutralizing antibody response, and our data suggest timing and/or superinfecting viral envelope characteristics as contributing factors. These results highlight differential outcomes in intrasubtype superinfection and provide the first insight into cases with CRF02_AG, the fourth most prevalent HIV-1 strain worldwide.