Use of a Chinese hamster ovary cell cytotoxicity assay for the rapid diagnosis of pertussis.

A cytotoxicity assay with Chinese hamster ovary cells (CHO) capable of detecting 750 pg of pertussis toxin was assessed for use as a rapid test for the diagnosis of pertussis and compared with direct immunofluorescence (DFA). With pure bacterial cultures and simulated clinical specimens, the CHO assay detected as few as two colonies of Bordetella pertussis; no cytotoxicity occurred with other respiratory tract microorganisms. Next, nasopharyngeal aspirate secretions and nasopharyngeal cultures harvested after 72 h of incubation from 57 culture-positive and 201 culture-negative patients were examined. The CHO assay with nasopharyngeal secretions was positive in 25 (45%) of 55 culture-positive cases; DFA was positive in 15 (26%) of 57 cases (P = 0.05). The CHO assay with 72-h culture washes was positive in 42 (75%) of 57 culture-positive cases (P less than 0.001 compared with DFA). The CHO assay was more specific than DFA; all five CHO-positive, culture-negative cases were confirmed as true positives by serologic or toxin neutralization assays. In contrast, only 4 (36%) of 11 DFA-positive, culture-negative cases were confirmed as pertussis by serologic methods (P = 0.03). Combining the CHO assay with culture significantly decreased the delay in laboratory diagnosis of pertussis (3.30 versus 4.54 days; P = 0.01). The CHO assay is a sensitive and specific assay for the rapid diagnosis of pertussis.     

Detection of antibodies inhibiting the ADP-ribosyltransferase activity of pertussis toxin in human serum.

Bordetella pertussis produces a protein virulence factor termed pertussis toxin. Many candidate pertussis vaccines are based on the rationale that an immune response that neutralizes the virulence activities of this toxin, which are thought to arise from its catalytic ADP-ribosyltransferase activity, would be beneficial. The report describes two methods that quantify the inhibition of this activity by human serum. One, termed a direct assay, involves an initial incubation of toxin with serum, a second incubation that activates the toxin, and a third incubation that measures the ADP-ribosyltransferase activity of the mixture. The other assay, termed a plate assay, involves immobilization of the toxin, exposure of the immobilized toxin to serum and washing of the plate, and then activation and assay of the toxin's ADP-ribosyltransferase activity. The plate assay may be more selective than the direct assay in terms of identifying antibodies that neutralize the toxin in vivo. Sera from controls, selected patients presenting with cough, and vaccinated infants were first analyzed by the direct assay. In contrast to sera from controls, sera from several of the patients and vaccinated infants strongly inhibited activity. Dose-response curves of inhibition were determined for samples from three vaccinated infants by both the direct and plate assays. One of the samples had a dose-response curve of a different shape and thus differed not only in titer but also in functional characteristics. A comparison of inhibition of ADP-ribosyltransferase activity and neutralization in a CHO cell assay indicated that there was incomplete agreement between the two assays. Taken together, these results indicate that measurement of inhibition of ADP-ribosyltransferase activity by human serum is practical and may be useful in the evaluation of responses to pertussis vaccines.     

Pertussis serology: assessment of IgG anti-PT ELISA for replacement of the CHO cell assay

Two types of serological assays are commonly used for the assessment of pertussis vaccine-induced antibodies; the Chinese hamster ovary cell (CHO cell) assay and the immunoglobulin G (IgG) anti pertussis toxin (PT) enzyme-linked immunosorbent assay (IgG anti-PT ELISA). Recently, both the techniques have been modified to improve performance with sera with interfering activity (CHO cell assay) or with heat-treated sera (IgG anti-PT ELISA). These two improved techniques were compared by the analysis of 100 individual serum samples from a previous clinical trial and 213 sera from a longitudinal serum collection from 20 Danish adults recently vaccinated with the Danish acellular pertussis vaccine. The comparison showed a significant linear correlation between the results of the two assays with a p-value of <0.0001 for the 100 individual samples. We, therefore, conclude that the improved IgG anti-PT ELISA can be used as a replacement for the often troublesome and time-consuming CHO cell assay for the measurement of vaccine-induced human antibodies to PT.     

The assay of gosling hepatitis virus and antibody by spermagglutination and spermagglutination-inhibition. II. Spermagglutination-inhibition

Spermagglutination by gosling hepatitis virus was specifically inhibited by sera from sick and convalescent goslings exposed to experimental and natural infection, by hyperimmune horse serum and by sera from vaccinated geese. When compared with passive protection of goslings, neutralization of egg infectivity and precipitation techniques, the spermagglutination-inhibition technique was found to be a highly sensitive serological procedure for antibody detection and assay.     

Production and purification of Bordetella pertussis toxin

Pertussis toxin (PT) is the major protective antigen of acellular pertussis vaccine (aP). We have established an optimal culture condition for the growth of B. pertussis and the production of PT in a laboratory scale fermentor. It was found that when the dissolved oxygen in medium was supplied with pure oxygen instead of air, the yield of PT was dramatically increased (i.e. from 2-3 mg/l using air to 8-10 mg/l using pure oxygen). PT was purified by affinity chromatography using hydroxyapatite and fetuin-sepharose columns. SDS-PAGE analysis and CHO cell clustering test showed that the purified PT was comparable to the reference PT in purity and biological activity. The purified PT could be detoxified by formaldehyde (d-PT). The results of CHO cell clustering neutralization assay and ELISA showed that the antibody induced by d-PT in mice was comparable to that induced by PT contained in a commercial DTaP. These results indicated that the immunogenicity of our d-PT was retained after the purification and detoxification procedures.     

Computational prediction and identification of dengue virus-specific CD4(+) T-cell epitopes

In this study, we tried to identify dengue virus-specific CD4(+) T-cell epitopes, which can induce PBMC (peripheral blood mononuclear cells) isolated from DF convalescent patients (dengue virus type 1 infection) to secrete IFN-gamma. PBMC of DF convalescent patients were stimulated in vitro with dengue virus-derived peptides, which were prepared based on the prediction of dengue virus-specific CD4(+) T-cell epitopes by using RANKpep online software. Subsequently, the frequency of IFN-gamma producing T cells and percentage of IFN-gamma(+) CD4(+) T cells were measured by using ELISPOT assay and ICS assay (intracellular cytokine straining), respectively. The positive response of PBMC by ELISPOT showed that the numbers of SFC (spots forming cells) ranged from 50 to 310 SFC/1x10(6) PBMC. The positive response of PBMC by ICS assay showed that the percentage of IFN-gamma(+) CD4(+) T cells ranged from 0.03 to 0.27%. As a result, C(45-57) (KLVMAFIAFLRFL), E(396-408) (SSIGKMFEATARG), NS3(23-35) (YRILQRGLLGRSQ), and NS3(141-155) (NREGKIVGLYGNGVV) were identified as dengue virus-specific CD4(+) T-cell epitopes.     

Effect of monoclonal antibody to pertussis toxin on toxin activity.

Two distinct monoclonal antibodies, one to pertussis toxin subunit S2, called 9G8, and another to subunits S2 and S3, called 11E6, were generated from the hybridomas of myeloma SP2/0 and spleen cells of BALB/c mice immunized mainly with the subunit S234 complex. Binding ability of 9G8 and 11E6 to the subunits was confirmed by the enzyme-linked immunosorbent assay and immunoblotting analysis. Generation of 11E6 bound to both S2 and S3 might mean that there is common antigenicity between S2 and S3. Neutralizing activities of 9G8 and 11E6 on various biological activities of pertussis toxin, including ADP-ribosyltransferase and leukocytosis-promoting, islet-activating, permeability-increasing. Chinese hamster ovary (CHO) cell-clustering, and hemagglutinating activities, were compared with those of anti-S1 monoclonal antibodies 1B7 and 3F10, which were isolated and characterized in a previous study (H. Sato, A. Ito, J. Chiba, and Y. Sato, Infect. Immun. 46:422-428, 1984). 1B7 and 3F10 neutralized ADP-ribosyltransferase activity of pertussis toxin or S1, but 9G8 and 11E6 did not. 1B7 showed very potent neutralization against leukocytosis-promoting, islet-activating, permeability-increasing, and CHO cell-clustering activities of pertussis toxin, but 3F10 did not, although anti-ADP-ribosyltransferase activities of both antibodies were identical. 11E6 neutralized leukocytosis-promoting, islet-activating, CHO cell-clustering, and hemagglutinating activities but not permeability-increasing activity. 9G8 showed slight neutralization of leukocytosis-promoting and CHO cell-clustering activities. Specific activities of 1B7 and 11E6 in each neutralization test were higher than or almost comparable to those of polyclonal antibodies to pertussis toxin. The neutralizing mechanism of 1B7 and 11E6 in leukocytosis-promoting activity was compared. 11E6 seemed to interfere with the binding of pertussis toxin to receptors on mouse spleen cells.     

Resistance of recent measles virus wild-type isolates to antibody-mediated neutralization by vaccinees with antibody

The neutralization capacity of sera from Luxembourgian adolescent vaccinees and from Nigerian women with measles-induced immunity to a number of measles virus strains was compared. Although both cohorts were matched for their hemagglutination inhibition and standard neutralization titers, 12 of the 22 late convalescent sera, and only 6 of 24 vaccinees neutralized all viruses. Similarly, only 2 of 20 viruses were not neutralized by at least 75% of late convalescent sera, in comparison to 10 of 20 viruses that resisted neutralization by at least 75% of the vaccinees. The more resistant viruses were not limited to a certain clade. One Nigerian virus was resistant to neutralization by 30% of the late convalescent women and by 75% of vaccinees. These results suggest that qualitative differences in neutralizing antibodies may reduce further protection of infants by passively acquired immunity against wild-type viruses when vaccinated girls become mothers.     

Determination of pertactin IgG antibodies for the diagnosis of pertussis

Objective  To compare increases in serum IgG antibody against pertactin with increases in IgG against pertussis toxin and filamentous hemagglutinin (FHA) in non-vaccinated children, children vaccinated with pertussis toxoid, and adults, all with culture-confirmed pertussis.
Methods  During a double-blind, placebo-controlled, efficacy trial of a monocomponent pertussis toxoid vaccine, acute and convalescent sera were obtained from study children and family members with suspected pertussis. In the present study, IgG antibodies against pertactin, pertussis toxin and FHA (determined by ELISA) were compared in 207 individuals with culture-verified pertussis and paroxysmal cough for ≥ 21 days.
Results  Significant increases in geometric mean serum IgG against all antigens occurred in non-vaccinated children, but more children responded against pertussis toxin and FHA than against pertactin (96%, 97%, and 62%, respectively). Of the children who had pertussis even though they were vaccinated with the pertussis toxoid vaccine, 97% responded to FHA, while responses to pertussis toxin and pertactin were less common (68% and 61%, respectively). In the 20 adults, the proportions of responders to FHA, pertussis toxin and pertactin were 90%, 80% and 55%, respectively.
Conclusion  Determination of IgG against pertussis toxin and FHA in paired sera in non-vaccinated children with pertussis is a more sensitive diagnostic tool than determination of IgG against pertactin. Pertactin IgG determinations might be of value as a complement to the other antibody assays in vaccinated children and in adults.     

A serological survey on neutralizing antibody titer of SARS convalescent sera

A seroepidemiologic study was conducted in North China in 2003 to determine the neutralizing antibody titer of severe acute respiratory syndrome (SARS) convalescent sera. A total of 99 SARS convalescent serum samples were collected from patients from the Inner Mongolia Autonomous Region, Hebei Province, and Beijing 35-180 days after the onset of symptoms. The anti-SARS antibodies were detected by enzyme-linked immunosorbent assay (ELISA), neutralization assay, and Western blot. Eighty-seven serum samples were confirmed to be positive for SARS antibodies. The neutralizing antibody titer of the 87 positive sera was analyzed quantitatively by neutralization assay. The geometric mean titer (GMT) of the 87 convalescent sera was 1:61. The Kolmogorov-Smirnov test showed that the neutralizing antibody titers conform to normal distribution, which suggests that the average anti-SARS antibody level in this study was representative of the convalescent antibody level of the SARS population. This result could be useful for the development and quality control of SARS vaccines.     

Identification of Bordetella pertussis infection by shared-primer PCR.

A shared-primer PCR method for the detection of infection was developed by using primers derived from DNA sequences upstream of the structural genes for the porin proteins of Bordetella pertussis and Bordetella parapertussis. This method resulted in a 159-bp PCR product specific for B. pertussis and a 121-bp DNA fragment specific for B. parapertussis and allowed for the simultaneous detection of these pathogens. The PCR procedure was shown to be very specific since no PCR product was obtained from 36 non-Bordetella bacterial DNAs. Nasopharyngeal aspirates (NPAs) from children suspected of having pertussis were evaluated by the PCR method, culture, and the Chinese hamster ovary (CHO) cell assay, which detects pertussis toxin. B. pertussis was cultured from 119 of 205 NPAs assayed, and the presence of pertussis toxin was detected in 69 of the NPAs by the CHO cell assay. When ethidium bromide staining was used to detect PCR products, 100 NPAs gave positive results by shared-primer PCR; 94 of these NPAs were also positive by culture. The result indicated a sensitivity of 79% for PCR when culture was used as the standard. The sensitivity of PCR was increased to 95% when a digoxigenin immunoblot system was used. An additional 20 NPAs from patients with suspected pertussis that were culture negative also gave positive results by PCR. The specific and sensitive PCR method described here should be useful for both the clinical diagnosis of pertussis and case identification in vaccine trials.     

Monoclonal antibodies to pertussis toxin: utilization as probes of toxin function

Six monoclonal antibodies (MAbs) to pertussis toxin (PT) have been generated and characterized. Five of these MAbs (3CX4, 3C4D, 6D11C, 6FX1, and X2X5) interact with determinants on the catalytic subunit (S1) of PT, and one (6DX3) is specific for subunit S4. The MAbs are divided into three groups based upon their ability to neutralize the effects of PT in a Chinese hamster ovary (CHO) cell assay. Three of the MAbs (3CX4, 3C4D and 6D11C) had high neutralization titers, one MAb (6FX1) displayed weak neutralizing activity, and two MAbs (X2X5 and 6DX3) had no neutralizing ability. The combination of one of the high titer MAbs (3CX4) with the low titer MAb (6FX1) resulted in a synergistic enhancement of neutralizing capability. F(ab')2 fragments prepared from MAb's 3CX4 and X2X5 displayed activities in the CHO-cell assay which were identical to the native MAb's. The ability of the MAbs to neutralize PT in the CHO-cell toxin neutralization assay correlated with their ability to inhibit the in vitro ADP-ribosylation of PT. A competition ELISA method demonstrated that this panel of MAbs recognizes at least four separate epitopes on the PT molecule. Biotin-conjugated MAbs were shown to be useful reagents to probe the interaction of pertussis toxin with fetuin.     

Cross-clade neutralization patterns among HIV-1 strains from the six major clades of the pandemic evaluated and compared in two different models

A panel of paired primary virus isolates and envelope pseudoviruses from sixty strains representing six HIV-1 clades was tested for neutralization using pooled, clade-specific plasma in two prominently utilized neutralization platforms: a primary isolate assay using peripheral blood mononuclear cells (PBMC) and a pseudovirus assay using a reporter epithelial cell line. Using the PMBC assay, pairing of the antibody pool against homologous clade viruses generated the highest geometric mean neutralizing antibody titer in 4 out of 6 clades tested, and neutralization patterns showed numerous examples of reciprocal cross-recognition between antibody and viruses of specific clade pairs. In the pseudovirus assay, cross-clade neutralization was more limited, with fewer distinct cross-clade relationships evident. The clade C antibody pool was broadly cross-reactive, neutralizing the greatest number of viruses in both assays. These data highlight the importance of the neutralization assay format employed and suggest that clade C envelopes merit further evaluation for the elicitation of broadly neutralizing antibodies.     

Antibodies recognizing protective pertussis toxin epitopes are preferentially elicited by natural infection versus acellular immunization

Despite more than 50 years of vaccination, disease caused by the bacterium Bordetella pertussis persists, with rates increasing in industrialized countries over the past decade. This rise may be attributed to several factors, including increased surveillance, emergence of vaccine escape variants, waning immunity in adults, and the introduction of acellular subunit vaccines, which include chemically detoxified pertussis toxin (PTd). Two potently protective epitopes on pertussis toxin (PTx) are recognized by the monoclonal antibodies 1B7 and 11E6, which inhibit catalytic and cell-binding activities, respectively. In order to determine whether the PTx exposure route affects antibody responses to these epitopes, we analyzed sera from 30 adults with confirmed pertussis exposure and from 30 recently vaccinated adults for specific anti-PTx antibody responses and in vitro CHO cell neutralization titers. While overall titers against PTx and the genetically detoxified variant, PTg, containing the R9K and E129G substitutions, were similar in the two groups, titers against specific epitopes depended on the exposure route. Natural infection resulted in significantly higher titers of anti-PTx-subunit 1, 1B7-like, and 11E6-like antibodies, while acellular vaccination resulted in significantly higher titers of antibodies recognizing PTd. We also observed a correlation between in vitro protection and the presence of 1B7-like and 11E6-like antibodies. Notably, chemical detoxification, as opposed to genetic inactivation, alters the PTx tertiary and quaternary structure, thereby affecting conformational epitopes and recognition of PTx by 1B7 and 11E6. The lower levels of serum antibodies recognizing clinically relevant epitopes after vaccination with PTd support inclusion of PTg in future vaccines.     

Detection of foot-and-mouth disease virus-infected cattle by assessment of antibody response in oropharyngeal fluids.

The detection of foot-and-mouth disease virus (FMDV)-persistent carriers among convalescent ruminants is of paramount importance in the aftermath of a field outbreak. To this purpose, FMDV-specific antibody should be investigated first, since virus isolation procedures from such carriers are seriously constrained. The complexity of the overall picture may be compounded by possible emergency vaccinations in the affected areas at the beginning of the outbreak. In this case, it is suggested that mucosal rather than serum antibody be investigated. In fact, we showed that FMDV-infected cattle regularly mount an antibody response in oropharyngeal fluids, in contrast to vaccinated cattle. Antibody could be revealed by neutralization assays and/or an immunoglobulin A (IgA)-specific kinetic enzyme-linked immunosorbent assay (ELISA). Cattle vaccinated once seldom showed a mucosal antibody response, which could be only detected by a total immunoglobulin-specific kinetic ELISA. Very few, if any, cattle showed a mucosal IgA response after repeated vaccinations. Our kinetic, IgA-specific ELISA generally allowed an early detection of FMDV-infected cattle; in particular, it proved to be more sensitive than the usual indirect, antigen-trapping ELISA in experiments on saliva samples.     

Development of a peptide ELISA for discrimination between serological responses to equine herpesvirus type 1 and 4

A peptide-based enzyme-linked immunosorbent assay (ELISA) for discrimination between serological responses to equine herpesvirus type 1 (EHV-1) and 4 (EHV-4) was developed. Three and four peptides for EHV-1 and EHV-4, respectively, were designed and studied initially in the ELISA using sera from foals infected experimentally. The most promising peptide pair, derived from EHV-1 glycoprotein E and EHV-4 glycoprotein G, was evaluated further using acute and convalescent sera from horses infected experimentally and naturally as well as a panel of horse field sera. Ten pre- and post-vaccination serum pairs were similarly tested in the type-specific ELISA. The peptide ELISA was able to identify horses which had been infected with EHV-1 or EHV-4 as derived from the results using acute and convalescent sera collected from natural outbreaks. When applied to a set of field samples, the assay proved robust with respect to determining the EHV-1 and EHV-4 antibody status. Also, the peptide ELISA was able to detect type-specific seroconversion for EHV-1 in vaccinated animals. With further validation, the EHV-1/EHV-4 peptide ELISA described in this study could serve as a reliable and cost-effective alternative to current methods for serological EHV-1 and EHV-4 diagnosis.     

乙脑病毒prME蛋白与BALB/c鼠IgG Fc段编码基因联合构建DNA免疫研究

目的 研究IgG Fc编码基因对流行性乙型脑炎(Japanese encephalitis,JE)DNA疫苗免疫增强效应的影响.方法 巢式RT-PCR法从BALB/c鼠脾组织获取IgG Fc段编码基因,用限制性内切酶从含流行性乙型脑炎病毒(Japanese encephalitis virus,JEV)prME蛋白基因重组子获取prME蛋白基因,分别插入同-真核表达载体pcDNA3.1(+)不同酶切位点,构建蘑组子pJME/IgG Fc并经酶切及DNA测序分析.脂质体法将pJMrY/IgG Fc转染CHO细胞.免疫荧光、Western blot法检测转染的CHO细胞中融合蛋白分布与表达.将pJME/IgG Fc肌注免疫BALB/c鼠,检测小鼠脾特异性细胞毒T细胞(CTL)杀伤活性和中和抗体滴度.结果 pJME/IgG Fc经BamH Ⅰ/EcoR Ⅰ和BamH Ⅰ/Not Ⅰ酶切释出的插入子大小,(2001 bp,2730 bp)分别与预期结果相符合.所编码的融合蛋白相对分子质量(Mr)为101×103,主要分布于胞浆,少最分布于胞膜,pJME/IgG Fc转染CHO细胞经32次传代仍可表达融合蛋白.pJME/IgGFc免疫组中和抗体滴度与CTL活性较pJME及灭活疫苗组均升高(P<0.05).结论 pJME/IgG Fc成功构建,转染的CHO细胞可稳定表达融合蛋白,IgG Fc段编码基因能够增强JEV DNA疫苗的细胞和体液免疫应答.     

Development of an ELISA for detection of antibodies to avian reovirus in chickens

An enzyme-linked immunosorbent assay (ELISA) using the expressed sigmaC and sigmaB proteins which induce neutralizing antibodies as the coating antigen (sigmaC-sigmaB-ELISA) for the detection of antibodies to avian reovirus in chickens was developed and compared with serum neutralization and conventional ELISA tests. These assays were used to examine the sera from chickens vaccinated experimentally and farm chickens. The correlation rate between serum neutralization and a sigmaC-sigmaB-ELISA was 100% (156/156), and that between serum neutralization and conventional ELISA was 89.1% (139/156). The results revealed that preparation of an ELISA by using sigmaC and sigmaB of ARV as the coating antigen in detecting the field chicken sera in comparison with the conventional ELISA gave a titer more correlated to the serum neutralization test. The sigmaC-sigmaB-ELISA showed a higher correlation with the serum neutralization-positive and -negative sera than that obtained with conventional ELISA. This combination antigen may thus be the best suited for preparing an ELISA for improving the determination of the immune status of chicken flocks or for detection of chicken infections with avian reovirus.     

Contrasting human cytokine responses to promastigote whole-cell extract and the Leishmania analogue receptor for activated C kinase antigen of L. amazonensis in natural infection versus immunization

It is known that the same antigen can induce different immune responses, depending upon the way that it is presented to the immune system. The objective of this study was to compare cytokine responses of peripheral blood mononuclear cells (PBMC) from cutaneous leishmaniasis patients and subjects immunized with a first-generation candidate vaccine composed of killed Leishmania amazonensis promastigotes to a whole-cell promastigote antigen extract (La) and to the recombinant protein LACK (Leishmania analogue receptor for activated C kinase), both from L. amazonensis. Thirty-two patients, 35 vaccinees and 13 healthy subjects without exposure to Leishmania, were studied. Cytokine production was assessed by enzyme-linked immunosorbent assay and enzyme-linked immunospot assay. The interferon (IFN)-gamma levels stimulated by La were significantly higher and the levels of interleukin (IL)-10 significantly lower than those stimulated by LACK in the patient group, while LACK induced a significantly higher IFN-gamma production and a significantly lower IL-10 production compared with those induced by La in the vaccinated group. LACK also induced a significantly higher frequency of IFN-gamma-producing cells than did La in the vaccinated group. The contrast in the cytokine responses stimulated by LACK and La in PBMC cultures from vaccinated subjects versus patients indicates that the human immune response to crude and defined Leishmania antigens as a consequence of immunization differs from that induced by natural infection.     

SARS-CoV-2 evolves to reduce but not abolish neutralizing action

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants of concern (VOCs) have prolonged coronavirus disease 2019 (COVID-19) pandemic by escaping pre-existing immunity acquired by natural infection or vaccination. Elucidation of VOCs' mutation trends and evasion of neutralization is required to update current control measures. Mutations and the prevalence of VOCs were analyzed in the global immunization coverage rate context. Lentivirus-based pseudovirus neutralization analysis platforms for SARS-CoV-2 prototype strain (PS) and VOCs, containing Alpha, Beta, Gamma, Delta, and Omicron, were constructed based on the spike protein of each variant and HEK 293T cell line expressing the human angiotensin-converting enzyme 2 (hACE2) receptor on the surface, and an enhanced green fluorescent protein reporter. Serum samples from 65 convalescent individuals and 20 WIBP-CorV vaccine recipients and four therapeutic monoclonal antibodies (mAbs) namely imdevimab, casirivimab, bamlanivimab, and etesevimab were used to evaluate the neutralization potency against the variants. Pseudovirus-based neutralization assay platforms for PS and VOCs were established, and multiplicity of infection (MOI) was the key factor influencing the assay result. Compared to PS, VOCs may enhance the infectivity of hACE2-293T cells. Except for Alpha, other VOCs escaped neutralization to varying degrees. Attributed to favorable and emerging mutations, the current pandemic Omicron variant of all VOCs demonstrated the most significant neutralization-escaping ability to the sera and mAbs. Compared with the PS pseudovirus, Omicron had 15.7- and 3.71-fold decreases in the NT50 value (the highest serum dilution corresponding to a neutralization rate of 50%); and correspondingly, 90% and 43% of immunization or convalescent serum samples lost their neutralizing activity against the Omicron variant, respectively. Therefore, SARS-CoV-2 has evolved persistently with a strong ability to escape neutralization and prevailing against the established immune barrier. Our findings provide important clues to controlling the COVID-19 pandemic caused by new variants.