Assessment of safety and intranasal neutralizing antibodies of HPMC-based human anti-SARS-CoV-2 IgG1 nasal spray in healthy volunteers.

An HPMC-based nasal spray solution containing human IgG1 antibodies against SARS-CoV-2 (nasal antibody spray or NAS) was developed to strengthen COVID-19 management. NAS exhibited potent broadly neutralizing activities against SARS-CoV-2 with PVNT50 values ranging from 0.0035 to 3.1997 µg/ml for the following variants of concern (ranked from lowest to highest): Alpha, Beta, Gamma, ancestral, Delta, Omicron BA.1, BA.2, BA.4/5, and BA.2.75. Biocompatibility assessment showed no potential biological risks. Intranasal NAS administration in rats showed no circulatory presence of human IgG1 anti-SARS-CoV-2 antibodies within 120 h. A double-blind, randomized, placebo-controlled trial (NCT05358873) was conducted on 36 healthy volunteers who received either NAS or a normal saline nasal spray. Safety of the thrice-daily intranasal administration for 7 days was assessed using nasal sinuscopy, adverse event recording, and self-reporting questionnaires. NAS was well tolerated, with no significant adverse effects during the 14 days of the study. The SARS-CoV-2 neutralizing antibodies were detected based on the signal inhibition percent (SIP) in nasal fluids pre- and post-administration using a SARS-CoV-2 surrogate virus neutralization test. SIP values in nasal fluids collected immediately or 6 h after NAS application were significantly increased from baseline for all three variants tested, including ancestral, Delta, and Omicron BA.2. In conclusion, NAS was safe for intranasal use in humans to increase neutralizing antibodies in nasal fluids that lasted at least 6 h.

CHO-produced RBD-Fc subunit vaccines with alternative adjuvants generate immune responses against SARS-CoV-2.

Subunit vaccines feature critical advantages over other vaccine platforms such as stability, price, and minimal adverse effects. To maximize immunological protection of subunit vaccines, adjuvants are considered as main components that are formulated within the subunit vaccine. They can modulate adverse effects and enhance immune outcomes. However, the most suitable formulation providing the best immunological outcomes and safety are still under investigation. In this report, we combined recombinant RBD with human IgG1 Fc to create an RBD dimer. This fusion protein was expressed in CHO and formulated with alternative adjuvants with different immune activation including Montanide ISA51, Poly (I:C), and MPLA/Quil-A® as potential vaccine candidate formulations. Using the murine model, a potent induction of anti-RBD IgG antibodies in immunized mice sera were observed. IgG subclass analyses (IgG1/IgG2a) illustrated that all adjuvanted formulations could stimulate both Th1 and Th2-type immune responses in particular Poly (I:C) and MPLA/Quil-A®, eliciting greater balance. In addition, Montanide ISA51-formulated RBD-Fc vaccination provided a promising level of neutralizing antibodies against live wild-type SARS-CoV-2 in vitro followed by Poly (I:C) and MPLA/Quil-A®, respectively. Also, mice sera from adjuvanted formulations could strongly inhibit RBD:ACE2 interaction. This study offers immunogenicity profiles, forecasted safety based on Vaccine-associated enhanced disease (VAED) caused by Th1-skewed immunity, and neutralizing antibody analysis of candidates of RBD-Fc-based subunit vaccine formulations to obtain an alternative subunit vaccine formulation against SARS-CoV-2.

Immunogenicity and reactogenicity against the SARS-CoV-2 variants following heterologous primary series involving CoronaVac, ChAdox1 nCov-19 and BNT162b2 plus BNT162b2 booster vaccination: An open-label randomized study in healthy Thai adults.

We evaluated the immunogenicity and reactogenicity of heterologous COVID-19 primary schedules involving BNT162b2 (Pfizer-BioNTech), ChAdOx1 nCoV-19 (AstraZeneca) and CoronaVac (Sinovac) in healthy adults, as well as booster response to BNT162b2 following heterologous CoronaVac and ChAdOx1 nCoV-19 regimens. Participants were randomized to one of seven groups that received two-dose homologous BNT162b2 or heterologous combinations of CoronaVac, ChAdOx1 nCoV-19 and BNT162b2, with 4 weeks interval. A total of 210 participants were enrolled, 30 in each group. Median age of participants was 38 (19-60) years, and 108/210 (51.43%) were female. Overall adverse events after the second dose were mild to moderate. We found that groups that received BNT162b2 as second dose induced the highest anti-receptor binding domain IgG response against the ancestral strain [BNT162b2: geometric mean concentration (GMC) 2133-2249 BAU/mL; ChAdOx1 nCoV-19: 851-1201; CoronaVac: 137-225 BAU/mL], neutralizing antibodies (NAb) against Beta and Delta, and interferon gamma response. All groups induced low to negligible NAb against Omicron after second dose. A BNT162b2 booster (third dose) following heterologous CoronaVac and ChAdOx1 nCoV-19 regimens induced >140-fold increase in NAb titers against Omicron. Our findings indicate that heterologous regimens using BNT162b2 as the second dose may be an alternative schedule to maximize immune response. While heterologous two-dose schedules induced low NAb against Omicron, the use of an mRNA vaccine booster dose substantially increased the Omicron response. These findings are relevant for low-income countries considering heterologous primary and booster COVID-19 vaccine schedules.

Immunogenicity and safety of an intradermal ChAdOx1 nCoV-19 boost in a healthy population.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused a global pandemic. Two doses of an inactivated SARS-CoV-2 vaccine (CoronaVac) have been shown to be insufficient to protect against variants of concern (VOCs), while viral vector vaccines remain protective against the infection. Herein, we conducted a preliminary study to evaluate the safety and immunity in an adult population who received the conventional 2 dosage-regimen of inactivated SARS-CoV-2 vaccine; with an additional intradermal ChAdOx1 nCoV-19 reciprocal dosage (1:5). An Intramuscular ChAdOx1 nCoV-19 booster was also included as a control. Immediate and delayed local reactions were frequently observed in the fractional intradermal boost, but systemic side effects were significantly decreased compared to the conventional intramuscular boost. The anti-RBD-IgG levels, the neutralising function against delta variants, and T cell responses were significantly increased after boosting via both routes. Interestingly, the shorter interval elicited higher immunogenicity compared to the extended interval. Taken together, a reciprocal dosage of intradermal ChAdOx1 nCoV-19 booster reduces systemic adverse reactions and enhances non inferiority humoral and cellular immune responses compared to a full dose of intramuscular boosting. These findings provide for an effective vaccine management during the shortages of vaccine supply.

The STING Ligand and Delivery System Synergistically Enhance the Immunogenicity of an Intranasal Spike SARS-CoV-2 Vaccine Candidate.

The respiratory organ serves as a primary target site for SARS-CoV-2. Thus, the vaccine-stimulating immune response of the respiratory tract is significant in controlling SARS-CoV-2 transmission and disease development. In this study, mucoadhesive nanoparticles were used to deliver SARS-CoV-2 spike proteins (S-NPs) into the nasal tracts of mice. The responses in the respiratory organ and the systemic responses were monitored. The administration of S-NPs along with cGAMP conferred a robust stimulation of antibody responses in the respiratory tract, as demonstrated by an increase of IgA and IgG antibodies toward the spike proteins in bronchoalveolar lavages (BALs) and the lungs. Interestingly, the elicited antibodies were able to neutralize both the wild-type and Delta variant strains of SARS-CoV-2. Significantly, the intranasal immunization also stimulated systemic responses. This is evidenced by the increased production of circulating IgG and IgA, which were able to neutralize and bind specifically to the SARS-CoV-2 virion and spike protein. Additionally, this intranasal administration potently activated a splenic T cell response and the production of Th-1 cytokines, suggesting that this vaccine may well activate a cellular response in the respiratory tract. The results demonstrate that STING agonist strongly acts as an adjuvant to the immunogenicity of S-NPs. This platform may be an ideal vaccine against SARS-CoV-2.

The Pilot Study of Immunogenicity and Adverse Events of a COVID-19 Vaccine Regimen: Priming with Inactivated Whole SARS-CoV-2 Vaccine (CoronaVac) and Boosting with the Adenoviral Vector (ChAdOx1 nCoV-19) Vaccine.

In response to the SARS-CoV-2 Delta variant, which partially escaped the vaccine-induced immunity provided by two doses of vaccination with CoronaVac (Sinovac), the National Vaccine Committee recommended the heterologous CoronaVac-ChAdOx1 (Oxford−AstraZeneca), a prime−boost vaccine regimen. This pilot study aimed to describe the immunogenicity and adverse events of the heterologous CoronaVac-ChAdOx1 regimen, in comparison with homologous CoronaVac, and homologous ChAdOx1. Between May and August 2021, we recruited a total of 354 participants from four vaccination groups: the CoronaVac-ChAdOx1 vaccinee (n = 155), the homologous CoronaVac vaccinee (n = 32), the homologous ChAdOx1 vaccinee (n = 47), and control group of COVID-19 patients (n = 120). Immunogenicity was evaluated by measuring the level of IgG antibodies against the receptor-binding domain (anti-SRBD) of the SARS-CoV-2 spike protein S1 subunit and the level of neutralizing antibodies (NAbs) against variants of concern (VOCs) using the plaque reduction neutralization test (PRNT) and pseudovirus neutralization test (pVNT). The safety profile was recorded by interviewing at the 1-month visit after vaccination. The anti-SRBD level after the second booster dose of the CoronaVac-ChAdOx1 group at 2 weeks was higher than 4 weeks. At 4 weeks after the second booster dose, the anti-SRBD level in the CoronaVac-ChAdOx1 group was significantly higher than either homologous CoronaVac, the homologous ChAdOx1 group, and Control group (p < 0.001). In the CoronaVac-ChAdOx1 group, the PRNT50 level against the wild-type (434.5 BAU/mL) was the highest; followed by Alpha variant (80.4), Delta variant (67.4), and Beta variant (19.8). The PVNT50 level was also found to be at its highest against the wild-type (432.1); followed by Delta variants (178.3), Alpha variants (163.9), and Beta variant (42.2), respectively. The AEs in the CoronaVac-ChAdOx1 group were well tolerated and generally unremarkable. The CoronaVac-ChAdOx1 heterologous regimen induced higher immunogenicity and a tolerable safety profile. In a situation when only CoronaVac-ChAdOx1 vaccines are available, they should be considered for use in responding to the Delta variant.

Comparison of safety and immunogenicity of CoronaVac and ChAdOx1 against the SARS-CoV-2 circulating variants of concern (Alpha, Delta, Beta) in Thai healthcare workers.

Background: Inactivated vaccine (CoronaVac) and chimpanzee adenovirus-vector vaccine (ChAdOx1) have been widely used in resource-limited settings. However, the information on the reactogenicity and immunogenicity of these two vaccines in the same setting are limited. Methods: Healthy health care workers (HCWs) aged 18 years or older were randomly assigned to receive either two doses of CoronaVac at 4 weeks interval or two doses of ChAdOx1 at 10 weeks interval. Self-reported adverse events (AEs) were collected for 7 days following each vaccination. Immunogenicity was determined by IgG antibodies levels against receptor binding domain (RBD) of the SARS-CoV-2 spike protein (S1 subunit) and the 50% plaque reduction neutralization titers against various strains. Results: Of the 360 HCWs, 180 in each vaccine group, the median (interquartile range: IQR) age was 35 (29-44) years old and 84.2% were female. Participants who received ChAdOx1 reported higher frequency of AEs than those received CoronaVac after both the first dose (84.4% vs. 66.1%, P < 0.001) and second dose (75.6% vs. 60.6%, P = 0.002), with more AEs in those younger than 30 years of age for both vaccines. The seroconversion rates were 75.6% and 100% following the first dose of CoronaVac and ChAdOx1, respectively. All participants were seropositive at 2 weeks after the second dose. The anti-SARS-CoV-2 RBD IgG levels induced by CoronaVac was lower than ChAdOX1 with geometric means of 164.4 and 278.5 BAU/mL, respectively (P = 0.0066). Both vaccines induced similar levels of neutralizing antibodies against the Wuhan strain, with the titers of 337.4 and 331.2; however, CoronaVac induced significantly lower GMT against Alpha (23.1 vs. 92.5), Delta (21.2 vs. 69.7), and Beta (10.2 vs. 43.6) variants, respectively. Conclusion: CoronaVac induces lower measurable antibodies against circulating variants but with lower frequency of AEs than ChAdOx1. An earlier boosting to prevent breakthrough infections may be needed.

Immunogenicity and Safety of an Intradermal BNT162b2 mRNA Vaccine Booster after Two Doses of Inactivated SARS-CoV-2 Vaccine in Healthy Population.

Effective vaccine coverage is urgently needed to tackle the COVID-19 pandemic. Inactivated vaccines have been introduced in many countries for emergency usage, but have only provided limited protection. Heterologous vaccination is a promising strategy to maximise vaccine immunogenicity. Here, we conducted a phase I, randomised control trial to observe the safety and immunogenicity after an intradermal boost, using a fractional dosage (1:5) of BNT162b2 mRNA vaccine in healthy participants in Songkhla, Thailand. In total, 91 volunteers who had been administered with two doses of inactivated SARS-CoV-2 (CoronaVac) were recruited into the study, and then randomised (1:1:1) to received different regimens of the third dose. An intramuscular booster with a full dose of BNT162b2 was included as a conventional control, and a half dose group was included as reciprocal comparator. Both, immediate and delayed adverse events following immunisation (AEFI) were monitored. Humoral and cellular immune responses were examined to observe the booster effects. The intradermal booster provided significantly fewer systemic side effects, from 70% down to 19.4% (p < 0.001); however, they were comparable to local reactions with the conventional intramuscular booster. In the intradermal group after receiving only one fifth of the conventional dosage, serum Anti-RBD IgG was halved compared to the full dose of an intramuscular injection. However, the neutralising function against the Delta strain remained intact. T cell responses were also less effective in the intradermal group compared to the intramuscular booster. Together, the intradermal booster, using a fractional dose of BNT162b2, can reduce systemic reactions and provides a good level and function of antibody responses compared to the conventional booster. This favourable intradermal boosting strategy provides a suitable alternative for vaccines and effective vaccine management to increase the coverage during the vaccine shortage.

Peritoneal Administration of a Subunit Vaccine Encapsulated in a Nanodelivery System Not Only Augments Systemic Responses against SARS-CoV-2 but Also Stimulates Responses in the Respiratory Tract.

The COVID-19 pandemic has currently created an unprecedented threat to human society and global health. A rapid mass vaccination to create herd immunity against SARS-CoV-2 is a crucial measure to ease the spread of this disease. Here, we investigated the immunogenicity of a SARS-CoV-2 subunit vaccine candidate, a SARS-CoV-2 spike glycoprotein encapsulated in N,N,N-trimethyl chitosan particles or S-TMC NPs. Upon intraperitoneal immunization, S-TMC NP-immunized mice elicited a stronger systemic antibody response, with neutralizing capacity against SARS-CoV-2, than mice receiving the soluble form of S-glycoprotein. S-TMC NPs were able to stimulate the circulating IgG and IgA as found in SARS-CoV-2-infected patients. In addition, spike-specific T cell responses were drastically activated in S-TMC NP-immunized mice. Surprisingly, administration of S-TMC NPs via the intraperitoneal route also stimulated SARS-CoV-2-specific immune responses in the respiratory tract, which were demonstrated by the presence of high levels of SARS-CoV-2-specific IgG and IgA in the lung homogenates and bronchoalveolar lavages of the immunized mice. We found that peritoneal immunization with spike nanospheres stimulates both systemic and respiratory mucosal immunity.

Intranasal Administration of RBD Nanoparticles Confers Induction of Mucosal and Systemic Immunity against SARS-CoV-2.

Mucosal immunity plays a significant role in host defense against viruses in the respiratory tract. Because the upper respiratory airway is a primary site of SARS-CoV-2 entry, immunization at the mucosa via the intranasal route could potentially lead to induction of local sterilizing immunity that protects against SARS-CoV-2 infection. In this study, we evaluated the immunogenicity of a receptor-binding domain (RBD) of SARS-CoV-2 spike glycoprotein loaded into N,N,N-trimethyl chitosan nanoparticles (RBD-TMC NPs). We showed that intranasal delivery of RBD-TMC NPs into mice induced robust local mucosal immunity, as evidenced by the presence of IgG and IgA responses in BALs and the lungs of immunized mice. Furthermore, mice intranasally administered with this platform of immunogens developed robust systemic antibody responses including serum IgG, IgG1, IgG2a, IgA and neutralizing antibodies. In addition, these immunized mice had significantly higher levels of activated splenic CD4+ and CD8+ cells compared with those that were administered with soluble RBD immunogen. Collectively, these findings shed light on an alternative route of vaccination that mimics the natural route of SARS-CoV-2 infection. This route of administration stimulated not only local mucosal responses but also the systemic compartment of the immune system.

Plant-Produced Receptor-Binding Domain of SARS-CoV-2 Elicits Potent Neutralizing Responses in Mice and Non-human Primates.

The emergence of coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has affected global public health and economy. Despite the substantial efforts, only few vaccines are currently approved and some are in the different stages of clinical trials. As the disease rapidly spreads, an affordable and effective vaccine is urgently needed. In this study, we investigated the immunogenicity of plant-produced receptor-binding domain (RBD) of SARS-CoV-2 in order to use as a subunit vaccine. In this regard, RBD of SARS-CoV-2 was fused with Fc fragment of human IgG1 and transiently expressed in Nicotiana benthamiana by agroinfiltration. The plant-produced RBD-Fc fusion protein was purified from the crude extract by using protein A affinity column chromatography. Two intramuscular administration of plant-produced RBD-Fc protein formulated with alum as an adjuvant have elicited high neutralization titers in immunized mice and cynomolgus monkeys. Further it has induced a mixed Th1/Th2 immune responses and vaccine-specific T-lymphocyte responses which was confirmed by interferon-gamma (IFN-γ) enzyme-linked immunospot assay. Altogether, our results demonstrated that the plant-produced SARS-CoV-2 RBD has the potential to be used as an effective vaccine candidate against SARS-CoV-2. To our knowledge, this is the first report demonstrating the immunogenicity of plant-produced SARS-CoV-2 RBD protein in mice and non-human primates.

Effective equine immunization protocol for production of potent poly-specific antisera against Calloselasma rhodostoma, Cryptelytrops albolabris and Daboia siamensis.

Snake envenomation has been estimated to affect 1.8 million people annually with about 94,000 deaths mostly in poor tropical countries. Specific antivenoms are the only rational and effective therapy for these cases. Efforts are being made to produce effective, affordable and sufficient antivenoms for these victims. The immunization process, which has rarely been described in detail, is one step that needs to be rigorously studied and improved especially with regard to the production of polyspecific antisera. The polyspecific nature of therapeutic antivenom could obviate the need to identify the culprit snake species. The aim of this study was to produce potent polyspecific antisera against 3 medically important vipers of Thailand and its neighboring countries, namely Cryptelytrops albolabris "White lipped pit viper" (CA), Calleoselasma rhodostoma "Malayan pit viper" (CR), and Daboia siamensis "Russell's viper" (DS). Four horses were immunized with a mixture of the 3 viper venoms using the 'low dose, low volume multi-site' immunization protocol. The antisera showed rapid rise in ELISA titers against the 3 venoms and reached plateau at about the 8th week post-immunization. The in vivo neutralization potency (P) of the antisera against CA, CR and DS venoms was 10.40, 2.42 and 0.76 mg/ml, respectively and was much higher than the minimal potency limits set by Queen Soavabha Memorial Institute (QSMI). The corresponding potency values for the QSMI monospecific antisera against CA, CR and DS venoms were 7.28, 3.12 and 1.50 mg/ml, respectively. The polyspecific antisera also effectively neutralized the procoagulant, hemorrhagic, necrotic and nephrotoxic activities of the viper venoms. This effective immunization protocol should be useful in the production of potent polyspecific antisera against snake venoms, and equine antisera against tetanus, diphtheria or rabies.

CRF01_AE-specific neutralizing activity observed in plasma derived from HIV-1-infected Thai patients residing in northern Thailand: comparison of neutralizing breadth and potency between plasma derived from rapid and slow progressors.

BACKGROUND: Development of a protective vaccine against human immunodeficiency virus type 1 (HIV-1) is an important subject in the field of medical sciences; however, it has not yet been achieved. Potent and broadly neutralizing antibodies are found in the plasma of some HIV-1-infected patients, whereas such antibody responses have failed to be induced by currently used vaccine antigens. In order to develop effective vaccine antigens, it is important to reveal the molecular mechanism of how strong humoral immune responses are induced in infected patients. As part of such studies, we examined the correlation between the anti-HIV-1 neutralizing antibody response and disease progression. METHODOLOGY/PRINCIPAL FINDINGS: We evaluated the anti-HIV-1 neutralizing activity of plasma derived from 33 rapid and 34 slow progressors residing in northern Thailand. The level of neutralizing activity varied considerably among plasmas, and no statistically significant differences in the potency and breadth of neutralizing activities were observed overall between plasma derived from rapid and slow progressors; however, plasma of 4 slow progressors showed neutralizing activity against all target viruses, whereas none of the plasma of rapid progressors showed such neutralizing activity. In addition, 21% and 9% of plasmas derived from slow and rapid progressors inhibited the replication of more than 80% of CRF01_AE Env-recombinant viruses tested, respectively. Neutralization of subtype B and C Env-recombinant viruses by the selected plasma was also examined; however, these plasma samples inhibited the replication of only a few viruses tested. CONCLUSIONS/SIGNIFICANCE: Although no statistically significant differences were observed in the potency and breadth of anti-HIV-1 neutralizing activities between plasma derived from rapid and slow progressors, several plasma samples derived from slow progressors neutralized CRF01_AE Env-recombinant viruses more frequently than those from rapid progressors. In addition, plasma derived from HIV-1-infected Thai patients showed CRF01_AE-specific neutralizing activity.

Viral factors involved in adapter-related protein complex 2 alpha 1 subunit-mediated regulation of human immunodeficiency virus type 1 replication.

The presence of siRNA against adapter-related protein complex 2 alpha 1 subunit (AP2alpha) enhances human immunodeficiency virus type 1 (HIV-1) replication by up-regulating nuclear transport of viral genome. In this report, we examined possible viral factors involved in AP2alpha-mediated regulation of HIV-1 replication, namely, Gag matrix protein (MA), integrase (IN) and Vpr. Replication of mutant viruses lacking the nucleophilic property of one of these viral proteins was significantly enhanced by treating cells with AP2alpha siRNA, indicating that Gag MA, IN or Vpr is not specifically involved in AP2alpha-mediated enhancement of viral replication. In contrast, AP2alpha siRNA showed no effect on the level of gene transduction mediated by HIV-1-derived lentiviral vector (LV). Although virus-like LV particle and parental HIV-1 particle are composed of almost equivalent viral structural proteins, LV particles lack three accessory proteins, Vif, Vpr and Vpu, and a large portion of the HIV-1 genome. Vif, Vpr and Vpu were dispensable for AP2alpha siRNA-mediated enhancement of HIV-1 replication, indicating that a particular part of the HIV-1 genomic fragment deleted in the LV genome might be required for the enhancing effect of AP2alpha siRNA on viral replication. Taken together, these results suggest that an as yet undetermined gene fragment of the HIV-1 genome is involved in AP2alpha-mediated regulation of HIV-1 replication.

Host-specific genetic variation of highly pathogenic avian influenza viruses (H5N1).

The complete genome sequences of two isolates A/chicken/Egypt/CL6/07 (CL6/07) and A/duck/Egypt/D2br10/07 (D2br10/07) of highly pathogenic avian influenza virus (HPAI) H5N1 isolated at the beginning of 2007 outbreak in Egypt were determined and compared with all Egyptian HPAI H5N1 sequences available in the GenBank. Sequence analysis utilizing the RNA from the original tissue homogenate showed amino acid substitutions in seven of the viral segments in both samples. Interestingly, these changes were different between the CL6/07 and D2br10/07 when compared to other Egyptian isolates. Moreover, phylogenetic analysis showed independent sub-clustering of the two viruses within the Egyptian sequences signifying a possible differential adaptation in the two hosts. Further, pre-amplification analysis of H5N1 might be necessary for accurate data interpretation and identification of distinct factor(s) influencing the evolution of the virus in different poultry species.

The role of lysine residue at amino acid position 165 of human immunodeficiency virus type 1 CRF01_AE Gag in reducing viral drug susceptibility to protease inhibitors.

Recombinant human immunodeficiency virus type 1 (HIV-1) containing a CRF01_AE Gag, AE-Gag62, was significantly less susceptible to protease inhibitors (PIs) than the subtype B reference strain, NL4-3; therefore, the mechanism of how AE-Gag62 reduced viral drug susceptibility to PIs was studied in this report. The results showed that the lysine residue at amino acid position 165 (K165) of AE-Gag62 played a role in reducing the drug susceptibility of the recombinant virus to PIs. In addition, K165 potentially appears more frequently in CRF01_AE viruses than in the viruses of other major HIV-1 subtypes. Although K165 had no effect on the extent of recombinant protease-mediated in vitro Gag cleavage, it enhanced the incorporation of the Gag-Pol precursor protein, p160, into virions. Taken together, these results suggest that K165 of CRF01_AE Gag affects the regulation of virion assembly or maturation, and reduces viral drug susceptibility to PIs.

Design and evaluation of antiretroviral peptides corresponding to the C-terminal heptad repeat region (C-HR) of human immunodeficiency virus type 1 envelope glycoprotein gp41.

Two alpha-helical heptad repeats, N-HR and C-HR, located in the human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein gp41, play an important role in membrane fusion by forming a 6-helix bundle. C34, a peptide mimicking C-HR, inhibits the formation of the 6-helix bundle; thus, it has potential as a novel antiretroviral compound. In order to improve the inhibitory effect of C34 on HIV-1 replication, we designed new C34-derived peptides based on computational analysis of the stable conformation of the 6-helix bundle. Newly designed peptides showed a stronger inhibitory effect on the replication of recombinant viruses containing CRF01_AE, subtype B or subtype C Env than C34 or a fusion inhibitor, T-20. In addition, these peptides inhibited the replication of a T-20-resistant virus. We propose that these peptides could be applied to develop novel antiretroviral compounds to inhibit the replication of various subtypes of HIV-1 as well as of T-20-resistant variants.

The RING finger ubiquitin ligase RNF125/TRAC-1 down-modulates HIV-1 replication in primary human peripheral blood mononuclear cells.

CXCR4-using HIV-1 was previously shown to replicate more efficiently in a healthy donor-derived CD4(+) CD38(+) than in a CD4(+) CD38(-) T-cell subset after stimulation with interleukin (IL)-4. Here, we identified 3 cellular genes, which were expressed to a higher level in an IL-4-stimulated CD38(-) subset. One of the 3 genes, RNF125/TRAC-1, was involved in the down-regulation of HIV-1 replication not only in cell lines, but also in peripheral blood mononuclear cells. RNF125/TRAC-1 bears the RING finger domain, important for E3 ubiquitin protein ligase. Mutations in this domain of RNF125/TRAC-1 led to the loss of HIV-1 down-modulatory activity, suggesting that E3 ligase activity is necessary. In addition, the results of Northern blotting and reporter gene analysis indicated that RNF125/TRAC-1 function occurs at the viral transcription step. These results suggest that RNF125/TRAC-1 could function to recruit host factor(s) controlling HIV-1 transcription to the ubiquitin-proteasome pathway.

Determination of HIV type 1 CRF01_AE gag p17 and env-V3 consensus sequences for HIV/AIDS vaccine design.

A molecular epidemiological study of the gag p17 and env-V3 regions on HIV-infected drug users and blood donors was carried out in northern Thailand from 1998 through 2002 to determine the predominant subtype and consensus sequence (CS) for circulating HIV-1 strains. CRF01_AE was concluded to be a predominant strain and the nucleotide CSs in gag p17 and env-V3 showed only 1.26% and no difference from CS in the Los Alamos database, respectively. Our env-V3 CS was identical to the previously published CSs, suggesting that the CS was very conserved from 1990 through 2002 in Thailand. Gag p17 and env-V3 nucleotide sequences of seroconvertors in our subjects were quite similar to the CS and conserved for at least 9 and 6 years postinfection, respectively. These results suggest that the CS approach to the HIV-1 antigen design could overcome HIV diversity and help us develop an effective HIV/AIDS vaccine.