Non-neutralizing antibodies targeting the immunogenic regions of HIV-1 envelope reduce mucosal infection and virus burden in humanized mice.

Antibodies are principal immune components elicited by vaccines to induce protection from microbial pathogens. In the Thai RV144 HIV-1 vaccine trial, vaccine efficacy was 31% and the sole primary correlate of reduced risk was shown to be vigorous antibody response targeting the V1V2 region of HIV-1 envelope. Antibodies against V3 also were inversely correlated with infection risk in subsets of vaccinees. Antibodies recognizing these regions, however, do not exhibit potent neutralizing activity. Therefore, we examined the antiviral potential of poorly neutralizing monoclonal antibodies (mAbs) against immunodominant V1V2 and V3 sites by passive administration of human mAbs to humanized mice engrafted with CD34+ hematopoietic stem cells, followed by mucosal challenge with an HIV-1 infectious molecular clone expressing the envelope of a tier 2 resistant HIV-1 strain. Treatment with anti-V1V2 mAb 2158 or anti-V3 mAb 2219 did not prevent infection, but V3 mAb 2219 displayed a superior potency compared to V1V2 mAb 2158 in reducing virus burden. While these mAbs had no or weak neutralizing activity and elicited undetectable levels of antibody-dependent cellular cytotoxicity (ADCC), V3 mAb 2219 displayed a greater capacity to bind virus- and cell-associated HIV-1 envelope and to mediate antibody-dependent cellular phagocytosis (ADCP) and C1q complement binding as compared to V1V2 mAb 2158. Mutations in the Fc region of 2219 diminished these effector activities in vitro and lessened virus control in humanized mice. These results demonstrate the importance of Fc functions other than ADCC for antibodies without potent neutralizing activity.

Signal peptide of HIV-1 envelope modulates glycosylation impacting exposure of V1V2 and other epitopes.

HIV-1 envelope (Env) is a trimer of gp120-gp41 heterodimers, synthesized from a precursor gp160 that contains an ER-targeting signal peptide (SP) at its amino-terminus. Each trimer is swathed by ~90 N-linked glycans, comprising complex-type and oligomannose-type glycans, which play an important role in determining virus sensitivity to neutralizing antibodies. We previously examined the effects of single point SP mutations on Env properties and functions. Here, we aimed to understand the impact of the SP diversity on glycosylation of virus-derived Env and virus neutralization by swapping SPs. Analyses of site-specific glycans revealed that SP swapping altered Env glycan content and occupancy on multiple N-linked glycosites, including conserved N156 and N160 glycans in the V1V2 region at the Env trimer apex and N88 at the trimer base. Virus neutralization was also affected, especially by antibodies against V1V2, V3, and gp41. Likewise, SP swaps affected the recognition of soluble and cell-associated Env by antibodies targeting distinct V1V2 configurations, V3 crown, and gp41 epitopes. These data highlight the contribution of SP sequence diversity in shaping the Env glycan content and its impact on the configuration and accessibility of V1V2 and other Env epitopes.

Alterations of HIV-1 envelope phenotype and antibody-mediated neutralization by signal peptide mutations.

HIV-1 envelope glycoprotein (Env) mediates virus attachment and entry into the host cells. Like other membrane-bound and secreted proteins, HIV-1 Env contains at its N terminus a signal peptide (SP) that directs the nascent Env to the endoplasmic reticulum (ER) where Env synthesis and post-translational modifications take place. SP is cleaved during Env biosynthesis but potentially influences the phenotypic traits of the Env protein. The Env SP sequences of HIV-1 isolates display high sequence variability, and the significance of such variability is unclear. We postulate that changes in the Env SP influence Env transport through the ER-Golgi secretory pathway and Env folding and/or glycosylation that impact on Env incorporation into virions, receptor binding and antibody recognition. We first evaluated the consequences of mutating the charged residues in the Env SP in the context of infectious molecular clone HIV-1 REJO.c/2864. Results show that three different mutations affecting histidine at position 12 affected Env incorporation into virions that correlated with reduction of virus infectivity and DC-SIGN-mediated virus capture and transmission. Mutations at positions 8, 12, and 15 also rendered the virus more resistant to neutralization by monoclonal antibodies against the Env V1V2 region. These mutations affected the oligosaccharide composition of N-glycans as shown by changes in Env reactivity with specific lectins and by mass spectrometry. Increased neutralization resistance and N-glycan composition changes were also observed when analogous mutations were introduced to another HIV-1 strain, JRFL. To the best of our knowledge, this is the first study showing that certain residues in the HIV-1 Env SP can affect virus neutralization sensitivity by modulating oligosaccharide moieties on the Env N-glycans. The HIV-1 Env SP sequences thus may be under selective pressure to balance virus infectiousness with virus resistance to the host antibody responses. (289 words).

Seroprevalence of viral hepatitis A, B, C, D and E viruses in the Hormozgan province southern Iran.

BACKGROUND: Viral hepatitis is a global public health problem affecting millions of people worldwide, causing thousands of deaths due to acute and persistent infection, cirrhosis, and liver cancer. Providing updated serologic data can improve both surveillance and disease control programs. This study is aimed to determine the seroprevalence of markers for viral hepatitis (A, B, C, D and E) and the epidemiology of such infections in the general population of southern Iran's Hormozgan province. METHODS: Between 2016 and 2017, a total of 562 individuals with ages ranging from 1 to 86 years, who visited governmental public laboratories for routine check-ups, were tested for the presence of serological markers to hepatitis virus types A to E using enzyme-linked immunosorbent assays. RESULTS: The overall anti-hepatitis A virus (HAV) antibody seroprevalence was 93.2% (524/562). The prevalence of anti-hepatitis E virus (HEV) antibodies was 15.8% (89/562) among which 1.6% (9/562) of the seropositive individuals also had evidence of recent exposure to the virus (IgM positivity). Two and a half percent (14/562) were positive for hepatitis B surface (HBs) antigen, whereas 11.6% (65/562) tested positive for anti-hepatitis B core (HBc) antibodies. Among anti-HBc positive patients, 11% (7/65) had HBs Ag and 5% (3/65) were positive for anti-hepatitis D virus (HDV) antibodies. The prevalence of anti-hepatitis C virus (HCV) antibodies was 0.7% (4/562). The seroprevalence of anti-HAV, HEV IgG, anti-HBc antibodies, and HBs Ag increased with age. CONCLUSION: The present study confirms a high seroprevalence of HAV infection among the examined population and reveals high levels of endemicity for HEV in the region. Planned vaccination policies against HAV should be considered in all parts of Iran. In addition, improvements on public sanitation and hygiene management of drinking water sources for the studied area are recommended.

Signal peptide exchange alters HIV-1 envelope antigenicity and immunogenicity.

Introduction: HIV-1 envelope (Env) is the key target for antibodies (Abs) against the virus and thus an important HIV-1 vaccine component. Env is synthesized from a gp160 precursor with a signal peptide (SP) at its N-terminus. This study investigated the influence of the SP on Env antigenicity and immunogenicity. Methods: Env proteins from two HIV-1 isolates, AA05 and AC02, were analyzed as gp120 and gp160 in their native wild-type (WT) forms and as chimeras with swapped SPs (AA05-02 and AC02-05). The WT and chimeric Env were assessed for antigenicity and glycosylation using monoclonal antibodies (mAbs) and glycan probes. Immunogenicity was tested in mice using three vaccine types: gp120 protein, gp120 DNA+gp120 protein, and gp120 DNA+gp160 DNA. Results: The recombinant AC02 gp120 protein was antigenically superior to AA05 as indicated by higher reactivity with most mAbs tested. When SPs were swapped, the antigenicity of the chimeric gp120s (AA05-02 and AC02-05) resembled that of the gp120s from which the SPs were derived; AA05-02 was similar to AC02 and vice versa. Glycan probe reactivity followed a similar pattern: AA05-02 and AC02 showed similar affinity to high-mannose specific mAbs and lectins. Interestingly, the antigenicity of gp160s showed an opposite pattern; membrane-bound gp160 expressed with the AA05 SP (AA05 and AC02-05) showed greater mAb binding than gp160 with the AC02 SP (AC02 and AA05-02). Mice immunized with gp120 protein showed that AA05-02 induced stronger cross-reactive binding Ab responses than AA05 WT, and AC02 elicited stronger responses than AC02-05, indicating AC02 SP enhanced gp120 immunogenicity. However, when DNA vaccines were included (gp120 DNA+gp120 protein and gp120 DNA+gp160 DNA), the use of heterologous SPs diminished the immunogenicity of the WT immunogens. Among the three vaccine regimens tested, only gp120 DNA+gp160 DNA immunization elicited low-level Tier 2 neutralizing Abs, with AA05 WT inducing Abs with greater neutralization capabilities than AA05-02. Conclusion: These data demonstrate that the SP can significantly impact the antigenicity and immunogenicity of HIV-1 Env proteins. Hence, while SP swapping is a common practice in constructing Env immunogens, this study highlights the importance of careful consideration of the effects of replacing native SPs on the immunogenicity of Env vaccines.

Dual Role of HIV-1 Envelope Signal Peptide in Immune Evasion.

HIV-1 Env signal peptide (SP) is an important contributor to Env functions. Env is generated from Vpu/Env encoded bicistronic mRNA such that the 5' end of Env-N-terminus, that encodes for Env-SP overlaps with 3' end of Vpu. Env SP displays high sequence diversity, which translates into high variability in Vpu sequence. This study aimed to understand the effect of sequence polymorphism in the Vpu-Env overlapping region (VEOR) on the functions of two vital viral proteins: Vpu and Env. We used infectious molecular clone pNL4.3-CMU06 and swapped its SP (or VEOR) with that from other HIV-1 isolates. Swapping VEOR did not affect virus production in the absence of tetherin however, presence of tetherin significantly altered the release of virus progeny. VEOR also altered Vpu's ability to downregulate CD4 and tetherin. We next tested the effect of these swaps on Env functions. Analyzing the binding of monoclonal antibodies to membrane embedded Env revealed changes in the antigenic landscape of swapped Envs. These swaps affected the oligosaccharide composition of Env-N-glycans as shown by changes in DC-SIGN-mediated virus transmission. Our study suggests that genetic diversity in VEOR plays an important role in the differential pathogenesis and also assist in immune evasion by altering Env epitope exposure.

A cross-reactive mouse monoclonal antibody against rhinovirus mediates phagocytosis in vitro.

Rhinoviruses (RVs) are the main cause of the common cold worldwide. To date, more than 160 types of the virus have been recognized, categorized into three major species - A, B, and C. There are currently no approved vaccines available to prevent infection with RVs. To elicit antibodies against conserved regions located on capsid proteins of RV A viruses, mice were sequentially vaccinated with DNA plasmids encoding capsid proteins of different RV A types. After a final boost with whole virus, antibody-expressing hybridomas were generated. After isotyping, 11 monoclonal antibodies (mAbs) expressing an IgG subtype Fc-domain were selected for further expansion and purification. Three mAbs showed cross-reactivity against multiple strains of RV A viruses by ELISA, including strains A1A, A1B, A15, A16 and A49. Other mAbs had strain-specific binding patterns, with the majority of mAbs showing reactivity to RV-A15, the strain used for the final vaccination. We found that the RV-A15-specific mAbs, but not the cross-reactive mAbs, had neutralizing activity against RV-A15. An antibody dependent cellular phagocytosis (ADCP) assay revealed substantial ADCP activity for one of the cross-reactive mAbs. Epitope mapping of the neutralizing mAbs via escape mutant virus generation revealed a shared binding epitope on VP1 of RV-A15 for several neutralizing mAbs. The epitope of the ADCP-active, non-neutralizing mAb was determined by microarray analysis of peptides generated from the VP1 capsid protein. VP1-specific, cross-reactive antibodies, especially those with ADCP activity, could contribute to protection against RV infections.

Determining major genotypes of hepatitis C virus among transplant recipients by real-time polymerase chain reaction assay.

BACKGROUND: Hepatitis C virus (HCV) infection still exists as a health concern among the transplant patients. Because of the severity of the disease, different responses to treatment, and side effects resulting from long therapeutic period, determination of genotypes and viral loads can help choose the best treatment protocols. OBJECTIVES: This study aimed to determine the HCV genotypes and its distribution patterns among liver, kidney, and bone marrow recipient candidates across Iran, referred to Namazi Hospital, southern Iran. PATIENTS AND METHODS: A total of 101 individuals, including 44 (43.6%) liver, 55 (54.5%) kidney, and 2 (2%) bone marrow recipient candidates, with ages ranging between 5 and 74 years (Mean ±SD: 46.53 ± 13.73 y) participated in this study. From those, whole blood sample were collected and anti-HCV antibodies, RNA detection, and genotyping were performed on plasma using commercial chromatographic immunoassay, TaqMan one-step real-time polymerase chain reaction (RT-PCR), and genotyping RT-PCR kits, respectively. The frequencies of anti-HCV antibodies, RNA, various genotypes, and the viral load were compared with respect to gender, age, and transplant recipient groups. RESULTS: Of 101 individuals, 47 (46.5%) were positive for anti-HCV antibodies and 34 (33.7%) for RNA with a significant difference (P < 0.05). RNA copy number ranged from 4.6 × 103 to 3.11 × 107 copies/mL, median: 2.92 × 106 copies/mL, with no statistical differences in all groups. Analyses revealed no significant differences between the frequencies of anti-HCV antibodies or RNA in different groups. The frequencies of the genotypes 1 (50%) and 3 (35.3%) were higher than those of the genotypes 2 (2.9%), 4 (2.9%), and undetermined one (8.8%). Genotype 1 was significantly more prevalent in liver transplant recipients, those older than 40 years, and male cases (P < 0.05). CONCLUSIONS: Considering the high frequency of genotypes 1 and 3 among the studied groups, it is suggested that before and after transplantation programs be improved to manage and treat the disease efficiently, based on the standard protocols for such genotypes in the region. Accordingly, the occurrence of post-transplant complications due to immunosuppression among all the recipients as well as reinfection in HCV infected liver transplant patients can be diminished.