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The ongoing COVID-19 global pandemic has necessitated evaluating various disinfection technologies for reducing viral transmission in public settings. Ultraviolet (UV) radiation can inactivate pathogens and viruses but more insight is needed into the performance of different UV wavelengths and their applications. We observed greater than a 3-log reduction of SARS-CoV-2 infectivity with a dose of 12.5 mJ/cm2 of 254 nm UV light when the viruses were suspended in PBS, while a dose of 25 mJ/cm2 was necessary to achieve a similar reduction when they were in an EMEM culture medium containing 2%(v/v) FBS, highlighting the critical effect of media in which the virus is suspended, given that SARS-CoV-2 is always aerosolized when airborne or deposited on a surface. It was found that SARS-CoV-2 susceptibility (a measure of the effectiveness of the UV light) in a buffer such as PBS was 4.4-fold greater than that in a cell culture medium. Furthermore, we discovered the attenuation of UVC disinfection by amino acids, vitamins, and niacinamide, highlighting the importance of determining UVC dosages under a condition close to aerosols that wrap the viruses. We developed a disinfection model to determine the effect of the environment on UVC effectiveness with three different wavelengths, 222 nm, 254 nm, and 265 nm. An inverse correlation between the liquid absorbance and the viral susceptibility was observed. We found that 222 nm light was most effective at reducing viral infectivity in low absorbing liquids such as PBS, whereas 265 nm light was most effective in high absorbing liquids such as cell culture medium. Viral susceptibility was further decreased in N95 masks with 222 nm light being the most effective. The safety of 222 nm was also studied. We detected changes to the mechanical properties of the stratum corneum of human skins when the 222 nm accumulative exposure exceeded 50 J/cm2.The findings highlight the need to evaluate each UV for a given application, as well as limiting the dose to the lowest dose necessary to avoid unnecessary exposure to the public. [ABSTRACT FROM AUTHOR]

As part of the continuing effort to develop an effective HIV vaccine, we generated a poxviral vaccine vector (previously described) designed to improve on the results of the RV144 Phase III clinical trial. The construct, NYVAC-KC, is a replication-competent, attenuated recombinant of the vaccinia virus strain, NYVAC. NYVAC is a vector that has been used in many previous clinical studies but is replication deficient. Here we report a side-by-side comparison of replication-restricted NYVAC and replication-competent NYVAC-KC in a non-human primate study, which utilized a prime-boost regimen similar to that of RV144. NYVAC-C and NYVAC-C-KC express the HIV-1 antigens gp140 and Gag/Gag-Pol-Nef-derived VLPs from clade C and were used as the prime, with recombinant virus plus envelope protein as the boost. In nearly every T and B cell immune assay against HIV-1, including neutralization and antibody binding, NYVAC-C-KC induced a greater immune response than did NYVAC-C, indicating that replication competence in a poxvirus may improve upon the modestly successful regimen used in the RV144 clinical trial. [ABSTRACT FROM AUTHOR]

The use of heterologous immunization regimens and improved vector systems has led to increases in immunogenicity of HIV-1 vaccine candidates in nonhuman primates. In order to resolve interrelations between different delivery modalities, three different poxvirus boost regimens were compared. Three groups of rhesus macaques were each primed with the same DNA vaccine encoding Gag, Pol, Nef, and gp140. The groups were then boosted with either the vaccinia virus strain NYVAC or a variant with improved replication competence in human cells, termed NYVAC-KC. The latter was administered either by scarification or intramuscularly. Finally, macaques were boosted with adjuvanted gp120 protein to enhance humoral responses. The regimen elicited very potent CD4+ and CD8+ T cell responses in a well-balanced manner, peaking 2 weeks after the boost. T cells were broadly reactive and polyfunctional. All animals exhibited antigenspecific humoral responses already after the poxvirus boost, which further increased following protein administration. Polyclonal reactivity of IgG antibodies was highest against HIV-1 clade C Env proteins, with considerable cross-reactivity to other clades. Substantial effector functional activities (antibody-dependent cell-mediated cytotoxicity and antibody-dependent cell-mediated virus inhibition) were observed in serum obtained after the last protein boost. Notably, major differences between the groups were absent, indicating that the potent priming induced by the DNA vaccine initially framed the immune responses in such a way that the subsequent boosts with NYVAC and protein led only to an increase in the response magnitudes without skewing the quality. This study highlights the importance of selecting the best combination of vector systems in heterologous prime-boost vaccination regimens. [ABSTRACT FROM AUTHOR]

While as yet there is no vaccine against HIV/AIDS, the results of the phase III Thai trial (RV144) have been encouraging and suggest that further improvements of the prime/boost vaccine combination of a poxvirus and protein are needed. With this aim, in this investigation we have generated derivatives of the candidate vaccinia virus vaccine vector NYVAC with potentially improved functions. This has been achieved by the re-incorporation into the virus genome of two host range genes, K1L and C7L, in conjunction with the removal of the immunomodulatory viral molecule B19, an antagonist of type I interferon action. These novel virus vectors, referred to as NYVAC-C-KC and NYVAC-C-KC-DB19R, have acquired relevant biological characteristics, giving higher levels of antigen expression in infected cells, replication-competency in human keratinocytes and dermal fibroblasts, activation of selective host cell signal transduction pathways, and limited virus spread in tissues. Importantly, these replication-competent viruses have been demonstrated to maintain a highly attenuated phenotype. [ABSTRACT FROM AUTHOR]

Correct endoproteolytic maturation of gp160 is essential for the infectivity of human immunodeficiency virus type 1. This processing of human immunodeficiency virus-1 envelope protein, gp160, into gp120 and gp41 has been attributed to the activity of the cellular subtilisin-like proprotein convertase furin. The prototypic furin recognition cleavage site is Arg-X-Arg/Lys-Arg. Arg-Arg-Arg-Arg-Arg-Arg or longer iterations of polyarginine have been shown to be competitive inhibitors of substrate cleavage by furin. Here, we tested polyarginine for inhibition of productive human immunodeficiency virus-1-infection in T-cell lines, primary peripheral blood mononuclear cells, and macrophages. We found that polyarginine inhibited significantly human immunodeficiency virus-1 replication at concentrations that were benign to cell cultures ex vivo and mice in vivo. Using a fluorogenic assay, we demonstrated that polyarginine potently inhibited substrate-specific proteolytic cleavage by furin. Moreover, we verified that authentic processing of human immunodeficiency virus-1 gp160 synthesized in human cells from an infectious human immunodeficiency virus-1 (HIV-1) molecular clone was effectively blocked by polyarginine. Taken together, our data support that inhibitors of proteolytic processing of gp160 may be useful for combating human immunodeficiency virus-1 and that polyarginine represents a lead example of such inhibitors. [ABSTRACT FROM AUTHOR]

Nuclear factor-?B essential modulator (NEMO), also called IKK?, has been proposed as a ‘universal’ adaptor of the I-?B kinase (IKK) complex for stimuli such as proinflammatory cytokines, microbes, and the HTLV-I Tax oncoprotein. Currently, it remains unclear whether the many signals that activate NF-?B through NEMO converge identically or differently. We have adopted two approaches to answer this question. First, we generated and targeted intracellularly three NEMO-specific monoclonal antibodies (mAbs). These mAbs produced two distinct intracellular NF-?B inhibition profiles segregating TNFa from Tax activation. Second, using NEMO knockout mouse fibroblasts and 10 NEMO mutants, we found that different regions function in trans either to complement or to inhibit dominantly TNFa, IL-1ß, or Tax activation of NF-?B. For instance, NEMO (1-245 amino acids) supported Tax-mediated NF-?B activation, but did not serve TNFa- or IL-1ß signaling. Altogether, our findings indicate that while NEMO ‘universally’ adapts numerous NF-?B activators, it may do so through separable domains. We provide the first evidence that selective targeting of NEMO can abrogate oncogenic Tax signaling without affecting signals used for normal cellular metabolism.Oncogene (2003) 22, 8912-8923. doi:10.1038/sj.onc.1207058 [ABSTRACT FROM AUTHOR]

Full-length soluble HIV-1 Tat protein has been shown to bind the CXCR4 receptor. Occupancy of CXCR4 by Tat inhibits infection of cells by T-tropic HIV-1. To understand if fragments of the Tat protein may have similar anti-HIV activity, we synthesized Tat peptides and tested their activity in tissue culture. Here, we report a sequence-specific contribution of Tat residues 31–35 to anti-HIV-1 activity. [Copyright &y& Elsevier]

Editorial. Discusses researches on T-cells. Role of human T-cell leukemia virus type 1 on adult T-cell leukemia; Perspectives on how Tax protein might influence the cellular capacity to correct damaged DNA.

Background:Hepatitis C virus (HCV) coinfection was reported to negatively affect HIV disease and HIV infection has a deleterious effect on HCV-related liver disease. However, despite common occurrence of HCV/HIV coinfection little is known about the mechanisms of interactions between the two viruses. Methods:We studied CD4+ and CD8+ T cell and CD19+ B cell apoptosis in 104 HIV-positive patients (56 were also HCV-positive) and in 22 HCV/HIV-coinfected patients treated for chronic hepatitis C with pegylated interferon and ribavirin. We also analyzed HCV/HIV coinfection in a Daudi B-cell line expressing CD4 and susceptible to both HCV and HIV infection. Apoptosis was measured by AnnexinV staining. Results:HCV/HIV coinfected patients had lower CD4+ and CD8+ T cell apoptosis and higher CD19+ B cell apoptosis than those with HIV monoinfection. Furthermore, anti-HCV treatment of HCV/HIV coinfected patients was followed by an increase of CD4+ and CD8+ T cell apoptosis and a decrease of CD19+ B cell apoptosis. In the Daudi CD4+ cell line, presence of HCV infection facilitated HIV replication, however, decreased the rate of HIV-related cell death. Conclusion:In HCV/HIV coinfected patients T-cells were found to be destroyed at a slower rate than in HIV monoinfected patients. These results suggest that HCV is a molecular-level determinant in HIV disease. [ABSTRACT FROM AUTHOR]

Pathogen reduction is a viable approach to ensure the continued safety of the blood supply against emerging pathogens. However, the currently licensed pathogen reduction techniques are ineffective against non-enveloped viruses such as hepatitis A virus, and they introduce chemicals with concerns of side effects which prevent their widespread use. In this report, we demonstrate the inactivation of both enveloped and non-enveloped viruses in human plasma using a novel chemical-free method, a visible ultrashort pulsed laser. We found that laser treatment resulted in 2-log, 1-log, and 3-log reductions in human immunodeficiency virus, hepatitis A virus, and murine cytomegalovirus in human plasma, respectively. Laser-treated plasma showed ≥70% retention for most coagulation factors tested. Furthermore, laser treatment did not alter the structure of a model coagulation factor, fibrinogen. Ultrashort pulsed lasers are a promising new method for chemical-free, broad-spectrum pathogen reduction in human plasma. [ABSTRACT FROM AUTHOR]

Abstract: The peptide sequence AcNH–TEG–Glu-Aib-Trp-AibAib-Trp-AibAib-Ile-Asp–OH (1), designed to display the WWI epitope found near the C-terminus of gp41, an envelope glycoprotein decorating the surface of the HIV-1 virus, has been synthesized and proved to have a relevant content of helical conformation because of the presence of five α-aminoisobutyric acid (Aib) units. Three copies of it have been connected to a tripodal platform based on 2,4,6-triethylbenzene-1,3,5-trimethylamine. The tripodal template 2 is even more structured than 1 thus suggesting a significant interaction between the three sequences connected to the platform. Preliminary inhibition assays of HIV-mediated cell fusion indicated that while the single peptide 1 is inactive within the concentration range of our assay, when it is conjugated to the tripodal platform, it is moderately active. These promising results suggest that our approach constitute a valid alternative to those reported so far. [Copyright &y& Elsevier]

Abstract: Vaccinia virus (VACV) has been used more extensively for human immunization than any other vaccine. For almost two centuries, VACV was employed to provide cross-protection against variola virus, the causative agent of smallpox, until the disease was eradicated in the late 1970s. Since that time, continued research on VACV has produced a number of modified vaccines with improved safety profiles. Attenuation has been achieved through several strategies, including sequential passage in an alternative host, deletion of specific genes or genetic engineering of viral genes encoding immunomodulatory proteins. Some highly attenuated third- and fourth-generation VACV vaccines are now being considered for stockpiling against a possible re-introduction of smallpox through bioterrorism. Researchers have also taken advantage of the ability of the VACV genome to accommodate additional genetic material to produce novel vaccines against a wide variety of infectious agents, including a recombinant VACV encoding the rabies virus glycoprotein that is administered orally to wild animals. This review provides an in-depth examination of these successive generations of VACV vaccines, focusing on how the understanding of poxviral replication and viral gene function permits the deliberate modification of VACV immunogenicity and virulence. [Copyright &y& Elsevier]

Antiretroviral therapy to treat AIDS uses molecules that target the reverse transcriptase and protease enzymes of human immunodeficiency virus, type 1 (HIV-1). A major problem associated with these treatments, however, is the emergence of drug-resistant strains. Thus, there is a compelling need to find drugs against other viral targets. One such target is the interaction between Tat, an HIV-1 regulatory protein essential for viral replication, and trans-activation-responsive (TAR) RNA. Here we describe the design and synthesis of an encoded combinatorial library containing 39,304 unnatural small molecules. Using a rapid high through-put screening technology, we identified 59 compounds. Structure-activity relationship studies led to the synthesis of 19 compounds that bind TAR RNA with high affinities. In the presence of a representative Tat-TAR inhibitor (5 µM TR87), we observed potent and sustained suppression of HIV replication in cultured cells over 24 days. The same concentration of this inhibitor did not exhibit any toxicity in cell cultures or in mice. TR87 was also shown to specifically disrupt Tat-TAR binding in vitro and inhibit Tat-mediated transcriptional activation in vitro and in vivo, providing a strong correlation between its activities and inhibition of HIV-1 replication. These results provide a structural scaffold for further development of new drugs, alone or in combination with other drugs, for treatment of HIV-1-infected individuals. Our results also suggest a general strategy for discovering pharmacophores targeting RNA structures that are essential in progression of other infectious, inflammatory, and genetic diseases. [ABSTRACT FROM AUTHOR]

A preventive human immunodeficiency virus type 1 (HIV-1) vaccine is an essential part of the strategy to eradicate AIDS. A critical question is whether antibodies that do not neutralize primary isolate (tier 2) HIV-1 strains can protect from infection. In this study, we investigated the ability of an attenuated poxvirus vector (NYVAC) prime-envelope gp120 boost to elicit potentially protective antibody responses in a rhesus macaque model of mucosal simian-human immunodeficiency virus (SHIV) infection. NYVAC vector delivery of a group M consensus envelope, trivalent mosaic envelopes, or a natural clade B isolate B.1059 envelope elicited antibodies that mediated neutralization of tier 1 viruses, cellular cytotoxicity, and phagocytosis. None of the macaques made neutralizing antibodies against the tier 2 SHIV SF162P3 used for mucosal challenge. Significant protection from infection was not observed for the three groups of vaccinated macaques compared to unvaccinated macaques, although binding antibody to HIV-1 Env correlated with decreased viremia after challenge. Thus, NYVAC Env prime-gp120 boost vaccination elicited polyfunctional, nonneutralizing antibody responses with minimal protective activity against tier 2 SHIV mucosal challenge. IMPORTANCE The antibody responses that confer protection against HIV-1 infection remain unknown. Polyfunctional antibody responses correlated with time to infection in previous macaque studies. Determining the ability of vaccines to induce these types of responses is critical for understanding how to improve upon the one efficacious human HIV-1 vaccine trial completed thus far. We characterized the antibody responses induced by a NYVAC-protein vaccine and determined the protective capacity of polyfunctional antibody responses in an R5, tier 2 mucosal SHIV infection model. [ABSTRACT FROM AUTHOR]

African swine fever virus (ASFV) causes an acute hemorrhagic fever in domestic pigs, with high socioeconomic impact. No vaccine is available, limiting options for control. Although live attenuated ASFV can induce up to 100% protection against lethal challenge, little is known of the antigens which induce this protective response. To identify additional ASFV immunogenic and potentially protective antigens, we cloned 47 viral genes in individual plasmids for gene vaccination and in recombinant vaccinia viruses. These antigens were selected to include proteins with different functions and timing of expression. Pools of up to 22 antigens were delivered by DNA prime and recombinant vaccinia virus boost to groups of pigs. Responses of immune lymphocytes from pigs to individual recombinant proteins and to ASFV were measured by interferon gamma enzyme-linked immunosorbent spot (ELISpot) assays to identify a subset of the antigens that consistently induced the highest responses. All 47 antigens were then delivered to pigs by DNA prime and recombinant vaccinia virus boost, and pigs were challenged with a lethal dose of ASFV isolate Georgia 2007/1. Although pigs developed clinical and pathological signs consistent with acute ASFV, viral genome levels were significantly reduced in blood and several lymph tissues in those pigs immunized with vectors expressing ASFV antigens compared with the levels in control pigs. IMPORTANCE The lack of a vaccine limits the options to control African swine fever. Advances have been made in the development of genetically modified live attenuated ASFV that can induce protection against challenge. However, there may be safety issues relating to the use of these in the field. There is little information about ASFV antigens that can induce a protective immune response against challenge. We carried out a large screen of 30% of ASFV antigens by delivering individual genes in different pools to pigs by DNA immunization prime and recombinant vaccinia virus boost. The responses in immunized pigs to these individual antigens were compared to identify the most immunogenic. Lethal challenge of pigs immunized with a pool of antigens resulted in reduced levels of virus in blood and lymph tissues compared to those in pigs immunized with control vectors. Novel immunogenic ASFV proteins have been identified for further testing as vaccine candidates. [ABSTRACT FROM AUTHOR]

Vaccinia virus (VACV) encodes an innate immune evasion protein, E3, which contains an N-terminal Z-nucleic acid binding (Zα) domain that is critical for pathogenicity in mice. Here we demonstrate that the N terminus of E3 is necessary to inhibit an IFN-primed virusinduced necroptosis. VACV deleted of the Zα domain of E3 (VACVE3LΔ83N) induced rapid RIPK3-dependent cell death in IFN-treated L929 cells. Cell death was inhibited by the RIPK3 inhibitor, GSK872, and infection with this mutant virus led to phosphorylation and aggregation of MLKL, the executioner of necroptosis. In 293T cells, induction of necroptosis depended on expression of RIPK3 as well as the host-encoded Zα domain-containing DNA sensor, DAI. VACVE3LΔ83N is attenuated in vivo, and pathogenicity was restored in either RIPK3- or DAI-deficient mice. These data demonstrate that the N terminus of the VACV E3 protein prevents DAI-mediated induction of necroptosis. [ABSTRACT FROM AUTHOR]

Showing modest efficacy, the RV144 HIV-1 vaccine clinical trial utilized a non-replicating canarypox viral vector and a soluble gp120 protein boost. Here we built upon the RV144 strategy by developing a novel combination of a replicating, but highly-attenuated Vaccinia virus vector, NYVAC-KC, and plant-produced HIV-1 virus-like particles (VLPs). Both components contained the full-length Gag and a membrane anchored truncated gp41 presenting the membrane proximal external region with its conserved broadly neutralizing epitopes in the pre-fusion conformation. We tested different prime/boost combinations of these components in mice and showed that the group primed with NYVAC-KC and boosted with both the viral vectors and plant-produced VLPs have the most robust Gag-specific CD8 T cell responses, at 12.7% of CD8 T cells expressing IFN-γ in response to stimulation with five Gag epitopes. The same immunization group elicited the best systemic and mucosal antibody responses to Gag and dgp41 with a bias towards IgG1. [ABSTRACT FROM AUTHOR]

We compared the HIV-1-specific immune responses generated by targeting HIV-1 envelope protein (Env gp140) to either CD40 or LOX-1, two endocytic receptors on dendritic cells (DCs), in rhesus macaques primed with a poxvirus vector (NYVAC-KC) expressing Env gp140. The DC-targeting vaccines, humanized recombinant monoclonal antibodies fused to Env gp140, were administered as a boost with poly-ICLC adjuvant either alone or coadministered with the NYVAC-KC vector. All the DC-targeting vaccine administrations with poly-ICLC increased the low-level serum anti-Env IgG responses elicited by NYVAC-KC priming significantly more (up to a P value of 0.01) than in a group without poly-ICLC. The responses were robust and cross-reactive and contained antibodies specific to multiple epitopes within gp140, including the C1, C2, V1, V2, and V3, C4, C5, and gp41 immunodominant regions. The DC-targeting vaccines also elicited modest serum Env-specific IgA responses. All groups gave serum neutralization activity limited to tier 1 viruses and antibodydependent cytotoxicity responses (ADCC) after DC-targeting boosts. Furthermore, CD4+ and CD8+ T cell responses specific to multiple Env epitopes were strongly boosted by the DC-targeting vaccines plus poly-ICLC. Together, these results indicate that prime-boost immunization via NYVAC-KC and either anti-CD40.Env gp140/poly-ICLC or anti-LOX-1.Env gp140/poly-ICLC induced balanced antibody and T cell responses against HIV-1 Env. Coadministration of NYVAC-KC with the DC-targeting vaccines increased T cell responses but had minimal effects on antibody responses except for suppressing serum IgA responses. Overall, targeting Env to CD40 gave more robust T cell and serum antibody responses with broader epitope representation and greater durability than with LOX-1. [ABSTRACT FROM AUTHOR]

Monkeypox virus (MPXV) infection fails to activate the host anti-viral protein, PKR, despite lacking a full-length homologue of the vaccinia virus (VACV) PKR inhibitor, E3. Since PKR can be activated by dsRNA produced during a viral infection, we have analyzed the accumulation of dsRNA in MPXV-infected cells. MPXV infection led to less accumulation of dsRNA than VACV infection. Because in VACV infections accumulation of abnormally low amounts of dsRNA is associated with mutations that lead to resistance to the anti-poxvirus drug isatin beta-thiosemicarbazone (IBT), we investigated the effects of treatment of MPXV-infected cells with IBT. MPXV infection was eight-fold more resistant to IBT than wild-type vaccinia virus (wtVACV). These results demonstrate that MPXV infection leads to the accumulation of less dsRNA than wtVACV, which in turn likely leads to a decreased capacity for activation of the dsRNA-dependent host enzyme, PKR. [ABSTRACT FROM AUTHOR]

Cyanovirin-N (CV-N) is an antiviral lectin with potent activity against enveloped viruses, including HIV. The mechanism of action involves high affinity binding to mannose-rich glycans that decorate the surface of enveloped viruses. In the case of HIV, antiviral activity of CV-N is postulated to require multivalent interactions with envelope protein gp120, achieved through a pseudo-repeat of sequence that adopts two near-identical glycan-binding sites, and possibly involves a 3D-domain-swapped dimeric form of CV-N. Here, we present a covalent dimer of CV-N that increases the number of active glycan-binding sites, and we characterize its ability to recognize four glycans in solution. A CV-N variant was designed in which two native repeats were separated by the "nested" covalent insertion of two additional repeats of CV-N, resulting in four possible glycan-binding sites. The resulting Nested CV-N folds into a wild-type-like structure as assessed by circular dichroism and NMR spectroscopy, and displays high thermal stability with a Tm of 59 °C, identical to WT. All four glycan-binding domains encompassed by the sequence are functional as demonstrated by isothermal titration calorimetry, which revealed two sets of binding events to dimannose with dissociation constants Kd of 25 µM and 900 µM, assigned to domains B and B' and domains A and A' respectively. Nested CV-N displays a slight increase in activity when compared to WT CV-N in both an anti-HIV cellular assay and a fusion assay. This construct conserves the original binding specifityies of domain A and B, thus indicating correct fold of the two CV-N repeats. Thus, rational design can be used to increase multivalency in antiviral lectins in a controlled manner. [ABSTRACT FROM AUTHOR]

Improved antigenicity against HIV-1 envelope (Env) protein is needed to elicit vaccine-induced protective immunity in humans. Here we describe the first tests in non-human primates (NHPs) of Env gp140 protein fused to a humanized anti-LOX-1 recombinant antibody for delivering Env directly to LOX-1-bearing antigen presenting cells, especially dendritic cells (DC). LOX-1, or 1ectin-like oxidized low-density lipoprotein (LDL) receptor-1, is expressed on various antigen presenting cells and endothelial cells, and is involved in promoting humoral immune responses. The anti-LOX-1 Env gp140 fusion protein was tested for priming immune responses and boosting responses in animals primed with replication competent NYVAC-KC Env gp140 vaccinia virus. Anti-LOX-1 Env gp140 vaccination elicited robust cellular and humoral responses when used for either priming or boosting immunity. Co-administration with Poly ICLC, a TLR3 agonist, was superior to GLA, a TLR4 agonist. Both CD4+ and CD8+ Env-specific T cell responses were elicited by anti-LOX-1 Env gp140, but in particular the CD4+ T cells were multifunctional and directed to multiple epitopes. Serum IgG and IgA antibody responses induced by anti-LOX-1 Env gp140 against various gp140 domains were cross-reactive across HIV-1 clades; however, the sera neutralized only HIV-1 bearing sequences most similar to the clade C 96ZM651 Env gp140 carried by the anti-LOX-1 vehicle. These data, as well as the safety of this protein vaccine, justify further exploration of this DC-targeting vaccine approach for protective immunity against HIV-1. [ABSTRACT FROM AUTHOR]

The vaccinia virus (VACV) E3 protein has been shown to be important for blocking activation of the cellular innate immune system and allowing viral replication to occur unhindered. Mutation or deletion of E3L severely affects viral host range and pathogenesis. While the monkeypox virus (MPXV) genome encodes a homologue of the VACV E3 protein, encoded by the F3L gene, the MPXV gene is predicted to encode a protein with a truncation of 37 N-terminal amino acids. VACV with a genome encoding a similarly truncated E3L protein (VACV-E3LΔ37N) has been shown to be attenuated in mouse models, and infection with VACV-E3LΔ37N has been shown to lead to activation of the host antiviral protein kinase R pathway. In this report, we present data demonstrating that, despite containing a truncated E3 homologue, MPXV phenotypically resembles a wild-type (wt) VACV rather than VACV-E3LΔ37N. Thus, MPXV appears to contain a gene or genes that can suppress the phenotypes associated with an N-terminal truncation in E3. The suppression maps to sequences outside F3L, suggesting that the suppression is extragenic in nature. Thus, MPXV appears to have evolved mechanisms to minimize the effects of partial inactivation of its E3 homologue. [ABSTRACT FROM AUTHOR]

An effective HIV-1 vaccine must induce protective antibody responses, as well as CD4+ and CD8+ T cell responses, that can be effective despite extraordinary diversity of HIV-1. The consensus and mosaic immunogens are complete but artificial proteins, computationally designed to elicit immune responses with improved cross-reactive breadth, to attempt to overcome the challenge of global HIV diversity. In this study, we have compared the immunogenicity of a transmitted-founder (T/F) B clade Env (B.1059), a global group M consensus Env (Con-S), and a global trivalent mosaic Env protein in rhesus macaques. These antigens were delivered using a DNA prime-rNYVAC vector and Env protein boost vaccination strategy. While Con-S Env was a single sequence, mosaic immunogens were a set of three Envs optimized to include the most common forms of potential T cell epitopes. Both Con-S and mosaic sequences retained common amino acids encompassed by both antibody and T cell epitopes, and were central to globally circulating strains. Mosaics and Con-S Envs expressed as full-length proteins bound well to a number of neutralizing antibodies with discontinuous epitopes. Also both consensus and mosaic immunogens induced significantly higher IFN-γ ELISpot responses than B.1059 immunogen. Immunization with these proteins, particularly Con-S, also induced significantly higher neutralizing antibodies to viruses than B.1059 Env, primarily to Tier 1 viruses. Both Con-S and mosaics stimulated more potent CD8-T cell responses against heterologous Envs than did B.1059. Both antibody and cellular data from this study strengthen the concept of using in silico designed centralized immunogens for global HIV-1 vaccine development strategies. [ABSTRACT FROM AUTHOR]

The generation of vaccines against HIV/AIDS able to induce long-lasting protective immunity remains a major goal in the HIV field. The modest efficacy (31.2%) against HIV infection observed in the RV144 phase III clinical trial highlighted the need for further improvement of HIV vaccine candidates, formulation, and vaccine regimen. In this study, we have generated two novel NYVAC vectors, expressing HIV-1 clade C gp140(ZM96) (NYVAC-gp140) or Gag(ZM96)-Pol-Nef(CN54) (NYVAC-Gag-Pol-Nef), and defined their virological and immunological characteristics in cultured cells and in mice. The insertion of HIV genes does not affect the replication capacity of NYVAC recombinants in primary chicken embryo fibroblast cells, HIV sequences remain stable after multiple passages, and HIV antigens are correctly expressed and released from cells, with Env as a trimer (NYVAC-gp140), while in NYVAC-Gag-Pol-Nef-infected cells Gag-induced virus-like particles (VLPs) are abundant. Electron microscopy revealed that VLPs accumulated with time at the cell surface, with no interference with NYVAC morphogenesis. Both vectors trigger specific innate responses in human cells and show an attenuation profile in immunocompromised adult BALB/c and newborn CD1 mice after intracranial inoculation. Analysis of the immune responses elicited in mice after homologous NYVAC prime/NYVAC boost immunization shows that recombinant viruses induced polyfunctional Env-specific CD4 or Gag-specific CD8 T cell responses. Antibody responses against gp140 and p17/p24 were elicited. Our findings showed important insights into virus-host cell interactions of NYVAC vectors expressing HIV antigens, with the activation of specific immune parameters which will help to unravel potential correlates of protection against HIV in human clinical trials with these vectors. [ABSTRACT FROM AUTHOR]

Attenuated poxviruses are safe and capable of expressing foreign antigens. Poxviruses are applied in veterinary vaccination and explored as candidate vaccines for humans. However, poxviruses express multiple genes encoding proteins that interfere with components of the innate and adaptive immune response. This manuscript describes two strategies aimed to improve the immunogenicity of the highly attenuated, host-range restricted poxvirus NYVAC: deletion of the viral gene encoding type-I interferon-binding protein and development of attenuated replication-competent NYVAC. We evaluated these newly generated NYVAC mutants, encoding HIV-1 env, gag, pol and nef, for their ability to stimulate HIV-specific CD8 T-cell responses in vitro from blood mononuclear cells of HIV-infected subjects. The new vectors were evaluated and compared to the parental NYVAC vector in dendritic cells (DCs), RNA expression arrays, HIV gag expression and cross-presentation assays in vitro. Deletion of type-I interferon-binding protein enhanced expression of interferon and interferoninduced genes in DCs, and increased maturation of infected DCs. Restoration of replication competence induced activation of pathways involving antigen processing and presentation. Also, replication-competent NYVAC showed increased Gag expression in infected cells, permitting enhanced cross-presentation to HIV-specific CD8 T cells and proliferation of HIV-specific memory CD8 T-cells in vitro. The recombinant NYVAC combining both modifications induced interferon-induced genes and genes involved in antigen processing and presentation, as well as increased Gag expression. This combined replication-competent NYVAC is a promising candidate for the next generation of HIV vaccines. [ABSTRACT FROM AUTHOR]

Summary: In this study, we evaluated the efficacy of vaccinia virus (VACV) containing mutations in the E3L virulence gene to protect mice against a lethal poxvirus challenge after vaccination by scarification. VACV strains with mutations in the E3L gene had significantly decreased pathogenicity, even in immune deficient mice, yet retained the ability to produce a potent Th1-dominated immune response in mice after vaccination by scarification, while protecting against challenge with wild type, pathogenic VACV. Initial experiments were done using the mouse-adapted, neurovirulent Western Reserve (WR) strain of vaccinia virus. Testing of the full E3L deletion mutation in the Copenhagen and NYCBH strains of VACV, which are more appropriate for use in humans, produced similar results. These results suggest that highly attenuated strains of VACV containing mutations in E3L have the potential for use as scarification administered vaccines. [Copyright &y& Elsevier]

The dynamics of hepatitis C virus (HCV) quasi species in the E2 region may correlate with the course of infection after orthotopic liver transplantation (OLT). Complexity was higher in group 1 patients than in group 2 patients before OLT (P < .02) and at 1 week after OLT (P < .04). Diversity decreased in group 1 at transmission, as measured by either SE (P < .01) or MMS (P < .04). However, diversity increased in this group after OLT, as measured by SE (P < .03) ) or MMS (P < .02). FS at 1 year after OLT correlated with transmission changes, as measured by SE (re = 0.642, P < .0001) and MMS (r = 0.443, P < .04), and with post-OLT changes (for SE: r = -0.583, P < .01; for MMS: r = -0.536, P < .01). HCV complexity and diversity in the E2 region correlated with the severity of recurrence of HCV infection after OLT. Increased diversity of quasi species at transmission correlated with a higher FS at 1 year. However, increased diversity of quasi species in the post-OLT period correlated with a lower FS at 1 year. The dynamics of HCV quasi species in patients who undergo transplantation are predictive of outcome. [ABSTRACT FROM AUTHOR]

Hepatitis C virus (HCV) is the major etiological agent of blood-borne non-A non-B hepatitis and a leading cause of liver cirrhosis and hepatocellular carcinoma worldwide. HCV core protein is a multi-functional protein with regulatory functions in cellular transcription and virus-induced transformation and pathogenesis. Here we report on the identification of a bZIP nuclear transcription protein as an HCV core cofactor for transformation. This bZIP factor, designated LZIP, activates CRE-dependent transcription and regulates cell proliferation. Loss of LZIP function in NIH 3T3 cells triggers morphological transformation and anchorage-independent growth. We show that HCV core protein aberrantly sequesters LZIP in the cytoplasm, inactivates LZIP function and potentiates cellular transformation. Our findings suggest that LZIP might serve a novel cellular tumor suppressor function that is targeted by the HCV core. [ABSTRACT FROM AUTHOR]