HIV-1抗体蛋白印迹确认与核酸检测复核对比研究

应用病毒核酸载量法NASBA和HIV-1 RNA的巢式逆转录PCR(nested RT-PCR)法与HIV抗体蛋白印迹(WB)方法,对经过初筛的44例HIV-1抗体阳性标本进行了对照检测研究。发现了2例(gp160、p24)和1例(gp160g、p120p、66、p24)的特殊阳性样本,经NASBA法和该RT-PCR法核酸检测为阴性;WB确认的4例gp160阳性带、1例p24、p17阳性带和13例p24阳性带,经NASBA法和该RT-PCR法核酸检测也为阴性;而WB确认的其余全部带型的抗体阳性标本经过NASBA法和该RT-PCR法检测均为阳性。该研究表明对只有gp160p、24和gp160、gp120p、66、p24的特殊阳性标本和以p24为主的抗体不确定标本需要用RT-PCR或NASBA方法进行核酸检测,以进一步确认。     

2017至2022年太原市HIV抗体不确定及后续随访阳转样本WB条带及流行病学因素分析

摘要 目的：探究HIV抗体不确定及后续随访阳转样本的WB条带和流行病学特征。方法：对太原市范围内2017年至2022年监测到的790份HIV抗体不确定及71例后续随访阳性人员的WB条带和流行病学信息进行分析。结果：（1）HIV抗体不确定样本主要来自医院、血液中心和自愿咨询与检测（VCT）机构,占比84.47%;阳转样本主要来自医院和VCT,占88.73%;不确定人群中的性病门诊就诊者和VCT人群阳转率最高,分别为50%、25.49%。（2）不确定和阳转样本以20-40周岁人员为主,占53.8%;不确定人群中的20-30岁阳转率最高,占15.57%。（3）不确定样本在学历、性别方面无明显差异,但阳转样本男性占比远高于女性、高学历人群相对较高。（4）不确定样本以已婚、无高危行为、就业人群为主,阳转样本以具有未婚、离异、丧偶、阳性配偶、男男同性恋（MSM）、学生、无业或待业等特点人群为主。（5）不确定样本条带以P24和P17条带和带型为主,阳转样本条带以P24和P17为主,以含有P24和gP160条带的带型为主。结论：不确定样本中具有性病门诊就诊者、VCT、MSM、20-30岁、未婚、无业等流行病学因素和具有P24或gP160条带等特点的样本后续阳转可能性较大,应以上述人群为防控干预重点,重点加强具有上述特点不确定样本的个案跟踪和随访检测。     

Mycobacterium tuberculosis Infection Interferes with HIV Vaccination in Mice

Tuberculosis (TB) has emerged as the most prominent bacterial disease found in human immunodeficiency virus (HIV)-positive individuals worldwide. Due to high prevalence of asymptomatic Mycobacterium tuberculosis (Mtb) infections, the future HIV vaccine in areas highly endemic for TB will often be administrated to individuals with an ongoing Mtb infection. The impact of concurrent Mtb infection on the immunogenicity of a HIV vaccine candidate, MultiHIV DNA/protein, was investigated in mice. We found that, depending on the vaccination route, mice infected with Mtb before the administration of the HIV vaccine showed impairment in both the magnitude and the quality of antibody and T cell responses to the vaccine components p24Gag and gp160Env. Mice infected with Mtb prior to intranasal HIV vaccination exhibited reduced p24Gag-specific serum IgG and IgA, and suppressed gp160Env-specific serum IgG as compared to respective titers in uninfected HIV-vaccinated controls. Importantly, in Mtb-infected mice that were HIV-vaccinated by the intramuscular route the virus neutralizing activity in serum was significantly decreased, relative to uninfected counterparts. In addition mice concurrently infected with Mtb had fewer p24Gag-specific IFN-γ-expressing T cells and multifunctional T cells in their spleens. These results suggest that Mtb infection might interfere with the outcome of prospective HIV vaccination in humans.     

用蛋白印迹确认实验检查"人类免疫缺陷病毒抗体不确定"结果的分析

目的 探讨蛋白印迹实验(WB)“人类免疫缺陷病毒(HIV)抗体不确定”结果的特点、产生的原因、WB确认实验存在的问题及可能的改善措施。方法 归纳分析本实验室2004～2005年的WB检测结果为“HIV抗体不确定”者的人群分布特点、实验检测特点、受检者随访复检及抗体转归情况。结果 “HIV抗体不确定”者人员构成中相对健康的自愿咨询检测者、献血员以及孕产妇占50%,“HIV抗体不确定”者随访困难、复检率较低;WB实验存在假阳性、尤以P24带较为严重。结论 “HIV抗体不确定”结果与WB实验的假阳性有关,实验室应采取应对措施尽可能减少“HIV抗体不确定”及对结果进行准确解释。     

Incorporation of high levels of chimeric human immunodeficiency virus envelope glycoproteins into virus-like particles

The human immunodeficiency virus (HIV) envelope (Env) protein is incorporated into HIV virions or virus-like particles (VLPs) at very low levels compared to the glycoproteins of most other enveloped viruses. To test factors that influence HIV Env particle incorporation, we generated a series of chimeric gene constructs in which the coding sequences for the signal peptide (SP), transmembrane (TM), and cytoplasmic tail (CT) domains of HIV-1 Env were replaced with those of other viral or cellular proteins individually or in combination. All constructs tested were derived from HIV type 1 (HIV-1) Con-S DeltaCFI gp145, which itself was found to be incorporated into VLPs much more efficiently than full-length Con-S Env. Substitution of the SP from the honeybee protein mellitin resulted in threefold-higher chimeric HIV-1 Env expression levels on insect cell surfaces and an increase of Env incorporation into VLPs. Substitution of the HIV TM-CT with sequences derived from the mouse mammary tumor virus (MMTV) envelope glycoprotein, influenza virus hemagglutinin, or baculovirus (BV) gp64, but not from Lassa fever virus glycoprotein, was found to enhance Env incorporation into VLPs. The highest level of Env incorporation into VLPs was observed in chimeric constructs containing the MMTV and BV gp64 TM-CT domains in which the Gag/Env molar ratios were estimated to be 4:1 and 5:1, respectively, compared to a 56:1 ratio for full-length Con-S gp160. Electron microscopy revealed that VLPs with chimeric HIV Env were similar to HIV-1 virions in morphology and size and contained a prominent layer of Env spikes on their surfaces. HIV Env specific monoclonal antibody binding results showed that chimeric Env-containing VLPs retained conserved epitopes and underwent conformational changes upon CD4 binding.     

HIV Entry and Envelope Glycoprotein-mediated Fusion

HIV entry involves binding of the trimeric viral envelope glycoprotein (Env) gp120/gp41 to cell surface receptors, which triggers conformational changes in Env that drive the membrane fusion reaction. The conformational landscape that the lipids and Env navigate en route to fusion has been examined by biophysical measurements on the microscale, whereas electron tomography, x-rays, and NMR have provided insights into the process on the nanoscale and atomic scale. However, the coupling between the lipid and protein pathways that give rise to fusion has not been resolved. Here, we discuss the known and unknown about the overall HIV Env-mediated fusion process.     

核酸定量检测试验应用于HIV-1感染实验室诊断的探讨

目的:探讨核酸定量检测在HIV-1感染实验室诊断中的应用。方法:选取145例第四代抗原/抗体联合诊断筛查试验为阳性反应的血浆样本,分别用Western印迹和HIV-1核酸定量方法进行检测,综合对比分析2种方法检测结果。结果:Western印迹检出阳性样本120例,不确定样本17例,阴性样本8例;HIV-1核酸定量试验检出结果大于检测限样本131例,其中包括12例Western印迹不确定样本、2例Western印迹阴性样本;有3例Western印迹阳性样本用HIV-1核酸定量检测试验未能检出。结论:核酸定量检测试验对于HIV-1感染阳性样本是一种有效的实验室诊断方法;对HIV-1核酸定量检测结果为"TND"的样本,建议加做Western印迹或结合其他补充试验结果进行综合诊断。     

HIV p24 as Scaffold for Presenting Conformational HIV Env Antigens

Heterologous protein scaffolds engrafted with structurally defined HIV Env epitopes recognized by broadly neutralizing monoclonal antibodies (MAbs) represent a promising strategy to elicit broad neutralizing antibodies. In such regards, a protein scaffold based on the HIV p24 CA protein is a highly attractive approach, providing also Gag epitopes for eliciting HIV non-neutralizing protective antibodies and specific CD4(+) and CD8(+) T cell responses. In the present study, computational techniques were employed to verify the presence of acceptor sites for conformational HIV Env epitopes and, as proof of concept, the analysis of HIV p24 CA-based scaffolds using a complete V3 loop in a MAb-bound conformation is presented. The V3-p24 epitope-scaffold proteins show the formation of capsomers made of hexamers similarly to the p24 wild type protein. Moreover, the conformational V3 loop presented on p24 scaffold is recognized by a panel of anti-V3 MAbs. The results suggest that HIV p24 CA protein has suitable acceptor sites for engrafting foreign epitopes, without disrupting the formation of capsomer hexamer structures, and that the V3 epitope does retain its antibody-bound conformation. This strongly support the feasibility of developing a scaffolding strategy based on p24 CA proteins displaying conformational minimal structural, antigenic HIV Env epitopes.     

Human Ubc9 Is Involved in Intracellular HIV-1 Env Stability after Trafficking out of the Trans-Golgi Network in a Gag Dependent Manner

The cellular E2 Sumo conjugase, Ubc9 interacts with HIV-1 Gag, and is important for the assembly of infectious HIV-1 virions. In the previous study we demonstrated that in the absence of Ubc9, a defect in virion assembly was associated with decreased levels of mature intracellular Envelope (Env) that affected Env incorporation into virions and virion infectivity. We have further characterized the effect of Ubc9 knockdown on HIV Env processing and assembly. We found that gp160 stability in the endoplasmic reticulum (ER) and its trafficking to the trans-Golgi network (TGN) were unaffected, indicating that the decreased intracellular mature Env levels in Ubc9-depleted cells were due to a selective degradation of mature Env gp120 after cleavage from gp160 and trafficked out of the TGN. Decreased levels of Gag and mature Env were found to be associated with the plasma membrane and lipid rafts, which suggest that these viral proteins were not trafficked correctly to the assembly site. Intracellular gp120 were partially rescued when treated with a combination of lysosome inhibitors. Taken together our results suggest that in the absence of Ubc9, gp120 is preferentially degraded in the lysosomes likely before trafficking to assembly sites leading to the production of defective virions. This study provides further insight in the processing and packaging of the HIV-1 gp120 into mature HIV-1 virions.     

HLA-binding regions of HIV-1 proteins. II. A systematic study of viral proteins.

To detect HLA-binding peptides in 10 HIV-1 proteins (Rev, Tat, Vif, Vpr, Vpu, Gag p18, Gag p24, Gag p15, Env gp120 and Env gp41), the peptide binding assay (PBA) has been performed using three HLA class I molecules. Correlations have been searched between the PBA results and the peptide competitor activity in a functional test using HLA-A2-restricted CTL and target cells. A correlation between the data found in the PBA and well-defined CTL epitopes could be attempted only for the three Gag proteins. For these proteins, our results are in agreement with the known existence of epitopes reacting with human CD8+ CTL, with some exceptions. Together with the results reported with a panel of Nef peptides, these experiments showed that at least 18/20 of the already reported CTL epitopes from HIV-1 Gag, Nef, and Env proteins could be detected by the PBA, most (17/18) corresponding to strong reactivities. Perhaps more important, the regions of HIV-1 Gag p24 or Nef proteins that contain multiple associated CTL epitopes, with different HLA restrictions, were clearly identified by the reactivities in the PBA of several overlapping peptides and the major practical interest of the PBA might be the detection of such polyepitopic regions. Prediction are proposed in this report for 10 proteins, including several proteins for which CTL epitopes remain presently unknown.     

TIP47 is Required for the Production of Infectious HIV‐1 Particles from Primary Macrophages

Macrophages are among the major targets of HIV‐1 infection and play a key role in viral pathogenesis. Identification of the cellular cofactors involved in the production of infectious HIV‐1 from macrophages is thus crucial. Here, we investigated the role of the cellular cofactor TIP47 in HIV‐1 morphogenesis in primary macrophages. Using siRNA approach, we show that TIP47 is essential for HIV‐1 infectivity and propagation. TIP47 silencing disrupts Gag and Env colocalization in macrophages. Moreover, mutations in HIV‐1 Gag or Env, which abolish interaction with TIP47, impair HIV‐1 propagation and infectivity preventing colocalization of Gag and Env, Gag and Env coimmunoprecipitation. Interestingly, disruption of Gag‐TIP47 interaction by matrix mutation or TIP47 depletion also causes Gag to localize in scattered dots in the vicinity of the plasma membrane of macrophages. Therefore, TIP47 is required for the encounter between Gag and Env, and thus for the generation of infectious HIV‐1 particles from primary macrophages.     

Bacterial expression and characterization of nine polypeptides encoded by segments of the envelope gene of human immunodeficiency virus

Nine envelope (Env) polypeptides, encoding different regions of HIV gp120 and gp41 Env proteins, and accounting for approx. 96% of the entire Env precursor glycoprotein complex (gp160) were expressed in Escherichia coli at levels ranging from approx. 2 to 20% of total cellular protein. The recombinant polypeptides were produced either as hybrid products fused to the cII gene fragment of the lambda vector or in an unfused form without interfering cII products. Partially purified protein fractions of each polypeptide were characterized serologically by Western-blot analysis against a panel of well characterized human immunodeficiency virus (HIV)-positive human reference sera. Most of the Env polypeptides were highly immunoreactive with anti-gp120/gp41 antibodies present in the sera of patients with acquired immunodeficiency syndrome (AIDS) and AIDS-related diseases, but the patterns of reactivity were different. These results demonstrate that some of the antigenic determinants residing on the viral gp160 complex are retained on the surfaces of the recombinant Env polypeptides, and suggest that these sites are differentially immunogenic. These results are therefore interpreted in the context of an ongoing process towards using bacterially expressed HIV Env polypeptides to help define biological and structural epitopes to aid in the development of more sensitive diagnostic and therapeutic reagents in the fight against AIDS.     

Regulation of human immunodeficiency virus type 1 Env-mediated membrane fusion by viral protease activity

We and others have presented evidence for a direct interaction between the matrix (MA) domain of the human immunodeficiency virus type 1 (HIV-1) Gag protein and the cytoplasmic tail of the transmembrane envelope (Env) glycoprotein gp41. In addition, it has been postulated that the MA domain of Gag undergoes a conformational change following Gag processing, and the cytoplasmic tail of gp41 has been shown to modulate Env-mediated membrane fusion activity. Together, these results raise the possibility that the interaction between the gp41 cytoplasmic tail and MA is regulated by protease (PR)-mediated Gag processing, perhaps affecting Env function. To examine whether Gag processing affects Env-mediated fusion, we compared the ability of wild-type (WT) HIV-1 Env and a mutant lacking the gp41 cytoplasmic tail to induce fusion in the context of an active (PR(+)) or inactive (PR(-)) viral PR. We observed that PR(-) virions bearing WT Env displayed defects in cell-cell fusion. Impaired fusion did not appear to be due to differences in the levels of virion-associated Env, in CD4-dependent binding to target cells, or in the formation of the CD4-induced gp41 six-helix bundle. Interestingly, truncation of the gp41 cytoplasmic tail reversed the fusion defect. These results suggest that interactions between unprocessed Gag and the gp41 cytoplasmic tail suppress fusion.     

The matrix protein of human immunodeficiency virus type 1 is required for incorporation of viral envelope protein into mature virions.

Accumulating evidence suggests that the matrix (MA) protein of retroviruses plays a key role in virus assembly by directing the intracellular transport and membrane association of the Gag polyprotein. In this report, we show that the MA protein of human immunodeficiency virus type 1 is also critical for the incorporation of viral Env proteins into mature virions. Several deletions introduced in the MA domain (p17) of human immunodeficiency virus type 1 Gag polyprotein did not greatly affect the synthesis and processing of the Gag polyprotein or the formation of virions. Analysis of the viral proteins revealed normal levels of Gag and Pol proteins in these mutant virions, but the Env proteins, gp120 and gp41, were hardly detectable in the mutant virions. Our data suggest that an interaction between the viral Env protein and the MA domain of the Gag polyprotein is required for the selective incorporation of Env proteins during virus assembly. Such an interaction appears to be very sensitive to conformational changes in the MA domain, as five small deletions in two separate regions of p17 equally inhibited viral Env protein incorporation. Mutant viruses were not infectious in T cells. When mutant and wild-type DNAs were cotransfected into T cells, the replication of wild-type virus was also hindered. These results suggest that the incorporation of viral Env protein is a critical step for replication of retroviruses and can be a target for the design of antiviral strategies.     

Mutations in the matrix protein of human immunodeficiency virus type 1 inhibit surface expression and virion incorporation of viral envelope glycoproteins in CD4+ T lymphocytes.

Highly conserved amino acids in the second helix structure of the human immunodeficiency virus type 1 (HIV-1) MA protein were identified to be critical for the incorporation of viral Env proteins into HIV-1 virions from transfected COS-7 cells. The effects of these MA mutations on viral replication in the HIV-1 natural target cells, CD4+ T lymphocytes, were evaluated by using a newly developed system. In CD4+ T lymphocytes, mutations in the MA domain of HIV-1 Gag also inhibited the incorporation of viral Env proteins into mature HIV-1 virions. Furthermore, mutations in the MA domain of HIV-1 Gag reduced surface expression of viral Env proteins in CD4+ T lymphocytes. The synthesis of gp160 and cleavage of gp160 to gp120 were not significantly affected by MA mutations. On the other hand, the stability of gp120 in MA mutant-infected cells was significantly reduced compared to that in the parental wild-type virus-infected cells. These results suggest that functional interaction between HIV-1 Gag and Env proteins is not only critical for efficient incorporation of Env proteins into mature virions but also important for proper intracellular transport and stable surface expression of viral Env proteins in infected CD4+ T lymphocytes. A single amino acid substitution in MA abolished virus infectivity in dividing CD4+ T lymphocytes without significantly affecting virus assembly, virus release, or incorporation of Gag-Pol and Env proteins, suggesting that in addition to its functional role in virus assembly, the MA protein of HIV-1 also plays an important role in other steps of virus replication.     

Cross-Reactions between the Cytotoxic T-Lymphocyte Responses of Human Immunodeficiency Virus-Infected African and European Patients

The great variability of protein sequences from human immunodeficiency virus (HIV) type 1 (HIV-1) isolates represents a major obstacle to the development of an effective vaccine against this virus. The surface protein (Env), which is the predominant target of neutralizing antibodies, is particularly variable. Here we examine the impact of variability among different HIV-1 subtypes (clades) on cytotoxic T-lymphocyte (CTL) activities, the other major component of the antiviral immune response. CTLs are produced not only against Env but also against other structural proteins, as well as some regulatory proteins. The genetic subtypes of HIV-1 were determined for Env and Gag from several patients infected either in France or in Africa. The cross-reactivities of the CTLs were tested with target cells expressing selected proteins from HIV-1 isolates of clade A or B or from HIV type 2 isolates. All African patients were infected with viruses belonging to clade A for Env and for Gag, except for one patient who was infected with a clade A Env-clade G Gag recombinant virus. All patients infected in France were infected with clade B viruses. The CTL responses obtained from all the African and all the French individuals tested showed frequent cross-reactions with proteins of the heterologous clade. Epitopes conserved between the viruses of clades A and B appeared especially frequent in Gag p24, Gag p18, integrase, and the central region of Nef. Cross-reactivity also existed among Gag epitopes of clades A, B, and G, as shown by the results for the patient infected with the clade A Env-clade G Gag recombinant virus. These results show that CTLs raised against viral antigens from different clades are able to cross-react, emphasizing the possibility of obtaining cross-immunizations for this part of the immune response in vaccinated individuals.     

Photoinduced reactivity of the HIV-1 envelope glycoprotein with a membrane-embedded probe reveals insertion of portions of the HIV-1 Gp41 cytoplasmic tail into the viral membrane

The interactions of HIV-1 Env (gp120-gp41) with CD4 and coreceptors trigger a barrage of conformational changes in Env that drive the membrane fusion process. Various regions of gp41 have profound effects on HIV entry and budding. However, the precise interactions between gp41 and the membrane have not been elucidated. To examine portions of membrane proteins that are embedded in membrane lipids, we have studied photoinduced chemical reactions in membranes using the lipid bilayer specific probe iodonaphthyl azide (INA). Here we show that in addition to the transmembrane anchor, amphipatic sequences in the cytoplasmic tail (CT) of HIV-1 gp41 are labeled by INA. INA labeling of the HIV-1 gp41 CT was similar whether wild-type or a mutant HIV-1 was used with uncleaved p55 Gag, which does not allow entry. These results shed light on the disposition of the HIV-1 gp41 CT with respect to the membrane. Moreover, our data have general implications for topology of membrane proteins and their in situ interactions with the lipid bilayer.     

Mechanisms of alpha1-antitrypsin inhibition of cellular serine proteases and HIV-1 protease that are essential for HIV-1 morphogenesis

Proprotein processing is essential for HIV infectivity. Cellular trans-Golgi network (TGN) serine proteases (e.g., furin) are required to cleave HIV envelope gp160 to gp120. In addition, HIV protease (PR), an aspartyl protease, cleaves p55(Gag) to p24, etc., in budding virions. alpha1-Antitrypsin (alpha(1)AT) is cleaved by serine proteases, causing a conformational change in alpha(1)AT that sequesters and so inactivates the protease. alpha(1)AT blocks both gp160 and p55 processing, and so is a powerful inhibitor of HIV replication. We hypothesized that alpha(1)AT inhibited gp160 and p55 processing via different mechanisms, and that in both cases, alpha(1)AT bound and was itself cleaved by the proteases whose activities were blocked. alpha(1)AT delivered by SV(AT), a recombinant, Tag-deleted SV40-derived vector, localized to the TGN, co-precipitated with furin, and depleted furin from the TGN. After SV(AT) transduction and HIV challenge, alpha(1)AT was detected in resulting nascent immature HIV-1 virions. alpha(1)AT also blocked incorporation of the enzymatically active dimeric form of PR into HIV virions. Western analysis using recombinant proteins showed that alpha(1)AT directly bound HIV PR, and was cleaved by it. The simultaneous inhibition of two different steps in HIV morphogenesis both increases alpha(1)AT antilentiviral activity and decreases the possibility that HIV mutations will allow escape from inhibition.     

Protein-disulfide isomerase-mediated reduction of two disulfide bonds of HIV envelope glycoprotein 120 occurs post-CXCR4 binding and is required for fusion

The human immunodeficiency virus (HIV) envelope (Env) glycoprotein (gp) 120 is a highly disulfide-bonded molecule that attaches HIV to the lymphocyte surface receptors CD4 and CXCR4. Conformation changes within gp120 result from binding and trigger HIV/cell fusion. Inhibition of lymphocyte surface-associated protein-disulfide isomerase (PDI) blocks HIV/cell fusion, suggesting that redox changes within Env are required. Using a sensitive assay based on a thiol reagent, we show that (i) the thiol content of gp120, either secreted by mammalian cells or bound to a lymphocyte surface enabling CD4 but not CXCR4 binding, was 0.5-1 pmol SH/pmol gp120 (SH/gp120), whereas that of gp120 after its interaction with a surface enabling both CD4 and CXCR4 binding was raised to 4 SH/gp120; (ii) PDI inhibitors prevented this change; and (iii) gp120 displaying 2 SH/gp120 exhibited CD4 but not CXCR4 binding capacity. In addition, PDI inhibition did not impair gp120 binding to receptors. We conclude that on average two of the nine disulfides of gp120 are reduced during interaction with the lymphocyte surface after CXCR4 binding prior to fusion and that cell surface PDI catalyzes this process. Disulfide bond restructuring within Env may constitute the molecular basis of the post-receptor binding conformational changes that induce fusion competence.     

Evaluation of the Bio-Rad Geenius HIV 1/2 Confirmation Assay as an Alternative to Western Blot in the Korean Population: A Multi-Center Study

Recently updated recommendations for diagnosis of HIV infection suggest a new diagnostic algorithm including HIV-1/HIV-2 antibody differentiation immunoassay instead of western blot (WB) as a confirmatory testing. We evaluated Bio-Rad Geenius HIV1/2 confirmation assay as a simple and reliable alternative to WB in the Korean population with low HIV prevalence. The Geenius HIV1/2 was performed in a total of 192 serum specimens (140 reactive and 52 nonreactive specimens by ARCHITECT HIV Ag/Ab Combo assay) that were prospectively collected from five institutions. HIV-1 nucleic acid amplification test (NAT) was performed in negative or indeterminate specimens by Geenius HIV1/2 or WB. Among 140 reactive specimens by HIV Ag/Ab assay, 82 (58.6%) were positive for HIV-1 Ab by Geenius HIV1/2. Among 58 negative or indeterminate specimens by Geenius HIV1/2, four specimens (6.9%) were positive by HIV-1 NAT. The sensitivity and specificity of Geenius HIV1/2 were 95.3% and 100.0%, respectively. When we considered only WB, the sensitivity and specificity of Geenius HIV1/2 were 100.0% and 99.1%, respectively. Agreement between Geenius HIV1/2 and WB was excellent (weighted Kappa = 0.89). The Geenius HIV1/2 is simple and time-saving compared with WB. It has an excellent performance and can be a reliable alternative to WB. HIV-1 NAT should be performed in negative or indeterminate specimens by Geenius HIV1/2 to detect acute HIV infection as recommended in new HIV testing algorithms.