Antibodies to human immunodeficiency virus in human sera induce cell-mediated lysis of human immunodeficiency virus-infected cells

The capacity of human immunodeficiency virus (HIV) antibody-positive sera from homosexually active men without acquired immune deficiency syndrome to lyse the HIV-infected T cell lines MOLT-4f and CCRF-CEM (CEM) in cooperation with lymphocytes from normal donors was investigated. Twenty-seven HIV antibody-positive sera, most of which enhanced the killing of HIV-infected MOLT-4f and CEM target cells by normal mononuclear cells were studied in detail. HIV antibody-positive sera resulted in lysis at dilutions as high as 1/10,000. HIV antibody-negative sera did not augment lysis of infected target cells. In addition, lysis of uninfected targets was not enhanced in the presence of HIV antibody-positive sera. Because fractionation of the HIV antibody-positive sera on a protein A affinity column resulted in recovery of the activity from the IgG fraction, the extra cytotoxic activity mediated by nonimmune cells in the presence of immune sera appears to be antibody-dependent. Furthermore, the cytotoxic effector cells were in the nonrosetting fraction of lymphocytes and expressed Leu-11 (cluster designation (CD)15) antigens, which is characteristic of cells participating in antibody-dependent cellular cytotoxicity reactions. The antibody specificity of the sera, determined by radioimmunoprecipitation, provides evidence that antibody-dependent cellular cytotoxicity can occur even when there are no detectable antibodies directed against gag proteins. Sera which lacked detectable antibodies to the envelope protein gp120 by radioimmunoprecipitation did not mediate antibody-dependent cellular cytotoxicity.     

Assorted mutations in the envelope gene of simian immunodeficiency virus lead to loss of neutralization resistance against antibodies representing a broad spectrum of specificities

Simian immunodeficiency virus (SIV) of macaques isolate SIVmac239 is highly resistant to neutralization by polyclonal antisera or monoclonal antibodies, a property that it shares with most primary isolates of human immunodeficiency virus type 1 (HIV-1). This resistance is important for the ability of the virus to persist at high levels in vivo. To explore the physical features of the viral envelope complex that contribute to the neutralization-resistant phenotype, we examined a panel of SIVmac239 derivatives for sensitivity to neutralization by a large collection of monoclonal antibodies (MAbs). These MAbs recognize both linear and conformational epitopes throughout the viral envelope proteins. The variant viruses included three derivatives of SIVmac239 with substitutions in specific N-linked glycosylation sites of gp120 and a fourth variant that lacked the 100 amino acids that encompass the V1 and V2 loops. Also included in this study was SIVmac316, a variant of SIVmac239 with distributed mutations in env that confer significantly increased replicative capacity in tissue macrophages. These viruses were chosen to represent a broad range of neutralization sensitivities based on susceptibility to pooled, SIV-positive plasma. All three of these very different kinds of mutations (amino acid substitutions, elimination of N-glycan attachment sites, and a 100-amino-acid deletion spanning variable loops V1 and V2) dramatically increased sensitivity to neutralization by MAbs from multiple competition groups. Thus, the mutations did not simply expose localized epitopes but rather conferred global increases in neutralization sensitivity. The removal of specific N-glycan attachment sites from V1 and V2 led to increased sensitivity to neutralization by antibodies recognizing epitopes from both within and outside of the V1-V2 sequence. Surprisingly, while most of the mutations that gave rise to increased sensitivity were located in the N-terminal half of gp120 (surface subunit [SU]), the greatest increases in sensitivity were to MAbs recognizing the C-terminal half of gp120 or the ectodomain of gp41 (transmembrane subunit [TM]). This reagent set and information should now be useful for defining the physical, structural, thermodynamic, and kinetic factors that influence relative sensitivity to antibody-mediated neutralization.     

During readaptation in vivo, a tissue culture-adapted strain of feline immunodeficiency virus reverts to broad neutralization resistance at different times in individual hosts but through changes at the same position of the surface glycoprotein

The broad resistance to antibody-mediated neutralization of lentiviruses recently isolated from infected hosts is a poorly understood feature which might contribute to the ability of these viruses to persist and to the failure of experimental vaccines to protect against virulent viruses. We studied the underlying molecular mechanisms by examining the evolution of a neutralization-sensitive, tissue culture-adapted strain of feline immunodeficiency virus upon reinoculation into specific-pathogen-free cats. Reversion to broad neutralization resistance was observed in seven of seven inoculated animals and, in individual hosts, started to develop between less than 4 and more than 15 months from infection. After comparison of the envelope sequences of the inoculum virus, of an additional 4 neutralization-sensitive in vitro variants, and of 14 ex vivo-derived variants (6 neutralization sensitive, 5 resistant, and 3 with intermediate phenotype), a Lys-->Asn or -->Glu change at position 481 in the V4 region of the surface glycoprotein appeared as a key player in the reversion. This conclusion was confirmed by mutagenesis of molecularly cloned virus. Analysis of viral quasispecies and biological clones showed that the intermediate phenotype was due to transient coexistence of neutralization-sensitive and -resistant variants. Since the amino acid position involved was the same in four of four recent revertants, it is suggested that the number of residues that control reversion to broad neutralization resistance in FIV might be very limited. Amino acid 481 was found to be changed only in one of three putative long-term revertants. These variants shared a Ser-->Asn change at position 557 in region V5, which probably collaborated with other mutations in long-term maintenance of neutralization resistance, as suggested by the study of mutagenized virus.     

Passive immunotherapy in simian immunodeficiency virus-infected macaques accelerates the development of neutralizing antibodies

Passively transferred neutralizing antibodies can block lentivirus infection, but their role in postexposure prophylaxis is poorly understood. In this nonhuman-primate study, the effects of short-term antibody therapy on 5-year disease progression, virus load, and host immunity were explored. We reported previously that postinfection passive treatment with polyclonal immune globulin with high neutralizing titers against SIVsmE660 (SIVIG) significantly improved the 67-week health of SIVsmE660-infected Macaca mulatta macaques. Four of six treated macaques maintained low or undetectable levels of virus in plasma, compared with one of ten controls, while two rapid progressors controlled viremia only as long as the SIVIG was present. SIVIG treatment delayed the de novo production of envelope (Env)-specific antibodies by 8 weeks (13). We show here that differences in disease progression were also significant at 5 years postinfection, excluding rapid progressors (P = 0.05). Macaques that maintained 相似文献   

Increased neutralization sensitivity and reduced replicative capacity of human immunodeficiency virus type 1 after short-term in vivo or in vitro passage through chimpanzees

Development of disease is extremely rare in chimpanzees when inoculated with either T-cell-line-adapted neutralization-sensitive or primary human immunodeficiency virus type 1 (HIV-1), at first excluding a role for HIV-1 neutralization sensitivity in the clinical course of infection. Interestingly, we observed that short-term in vivo and in vitro passage of primary HIV-1 isolates through chimpanzee peripheral blood mononuclear cells (PBMC) resulted in a neutralization-sensitive phenotype. Furthermore, an HIV-1 variant reisolated from a chimpanzee 10 years after experimental infection was still sensitive to neutralization by soluble CD4, the CD4 binding site recognizing antibody IgG1b12 and autologous chimpanzee serum samples, but had become relatively resistant to neutralization by polyclonal human sera and neutralizing monoclonal antibodies. The initial adaptation of HIV-1 to replicate in chimpanzee PBMC seemed to coincide with a selection for viruses with low replicative kinetics. Neither coreceptor usage nor the expression level of CD4, CCR5, or CXCR4 on chimpanzee PBMC compared to human cells could explain the phenotypic changes observed in these chimpanzee-passaged viruses. Our data suggest that the increased neutralization sensitivity of HIV-1 after replication in chimpanzee cells may in part contribute to the long-term asymptomatic HIV-1 infection in experimentally infected chimpanzees.     

Association of structural changes in the V2 and V3 loops of the gp120 envelope glycoprotein with acquisition of neutralization resistance in a simian-human immunodeficiency virus passaged in vivo

The in vivo passage of a neutralization-sensitive, laboratory-adapted simian-human immunodeficiency virus (SHIV-HXBc2) generated a pathogenic, neutralization-resistant virus, SHIV-HXBc2P 3.2. SHIV-HXBc2P 3.2 differs from SHIV-HXBc2 only in 13 amino acid residues of the viral envelope glycoproteins. Here we used antibody competition analysis to examine the structural changes that occurred in the SHIV-HXBc2P 3.2 gp120 exterior envelope glycoprotein. The relationships among the antibody epitopes on the conserved gp120 core of SHIV-HXBc2 and SHIV-HXBc2P 3.2 were similar. The third variable (V3) loop was more closely associated with the fourth conserved (C4) region and CD4-induced epitopes on the gp120 core in the HXBc2P 3.2 gp120 glycoprotein compared with the HXBc2 gp120 glycoprotein. Rearrangements of the second variable (V2) loop with respect to the CD4 binding site and associated epitopes were evident in comparisons of the two gp120 glycoproteins. Thus, the in vivo evolution of a neutralization-resistant virus involves conformational adjustments of the V2 and V3 variable loops with respect to the conserved receptor-binding regions of the gp120 core.     

Human immunodeficiency virus type 1 primary isolate neutralization resistance is associated with the syncytium-inducing phenotype and lower CD4 cell counts in subtype CRF01_AE-infected patients

A number of human immunodeficiency virus type 1 (HIV-1) non-B-subtype products have been developed for present or future vaccine trials; in Thailand, several studies using subtype B and/or CRF01_AE vaccines have been conducted. To better characterize the biologic properties of these subtypes, 70 HIV-1 subtype B and E isolates were phenotyped as syncytium-inducing (SI) or non-syncytium-inducing (NSI) isolates and assessed for sensitivity to neutralizing antibody (NAb). A significantly higher number of NSI subtype E viruses were neutralization sensitive than SI subtype E viruses (P = 0.009), while no association between viral phenotype and sensitivity to NAb was observed for subtype B (P = 0.856), suggesting a difference in the neutralization patterns of subtypes B and E. Strikingly, concurrent CD4 T-cell numbers were significantly lower for subtype E-infected patients whose isolates were more resistant to NAb, both for the overall study group (P < 0.001) as well as for the 22 patients with NSI isolates (P = 0.013). Characterization of the evolution of biologic properties of both B and non-B HIV-1 subtypes will provide a clearer understanding of the repertoire of antibodies that must be elicited for a vaccine to be effective against all phenotypes and subtypes.     

絮状单位测定与小鼠体内中和试验法检测TAT效价的比较

目的为了更好地进行生产质量控制,快速准确地测定破伤风抗毒素(TAT)效价,以统计分析絮状单位测定法(体外法)和小鼠中和试验法(体内法)测定TAT效价的相关性。方法对2010—2013年破伤风抗毒素生产过程中的原料血浆、原液的絮状单位值与小鼠中和效价进行了统计分析和比较。结果体外法和体内法检测TAT原料血浆和原液效价的相关系数分别为r血浆=0.794 3,P0.001和r原液=0.901 6,P0.001,均呈显著性正相关。结论体外法可指导或替代体内法TAT效价测定应用于生产过程中间品的质量控制。     

Antibody-mediated in vitro neutralization of human immunodeficiency virus type 1 abolishes infectivity for chimpanzees.

This study was undertaken to establish whether antibody directed against the human immunodeficiency virus type 1 (HIV-1) principal gp120 type-specific neutralization determinant can abolish the infectivity of HIV-1 in chimpanzees. Challenge inocula of the IIIb virus isolate were mixed in vitro with either immunoglobulin G (IgG) from an uninfected chimpanzee, nonneutralizing IgG from an HIV-seropositive human, a virus-neutralizing murine monoclonal antibody directed against the HIV-1 IIIb isolate, or virus-neutralizing IgG from a chimpanzee infected with the IIIb isolate. Both neutralizing antibodies were directed against the principal neutralization determinant of the challenge isolate. Establishment of infection following inoculation of each virus-antibody mixture into chimpanzees was assessed by virus-specific antibody development and by virus isolation. No protective effect was noted either with the control IgG or with the nonneutralizing anti-HIV IgG. By contrast, the polyclonal chimpanzee virus-neutralizing IgG prevented HIV-1 in vivo infection, while the neutralizing monoclonal antibody notably decreased the infectivity of the challenge virus. Hence, antibody to the gp120 principal neutralization determinant is able both to prevent HIV-1 infection in vitro and to inhibit infection in vivo.     

Evidence for similar recognition of the conserved neutralization epitopes of human immunodeficiency virus type 1 envelope gp120 in humans and macaques

We compared the immune responses to the human immunodeficiency virus type 1 (HIV-1) envelope glycoproteins in humans and macaques with the use of clade A and clade B isogenic V3 loop glycan-possessing and -deficient viruses. We found that the presence or absence of the V3 loop glycan affects to similar extents immune recognition by a panel of anti-HIV human and anti-simian/human immunodeficiency virus (anti-SHIV) macaque sera. All sera tested neutralized the glycan-deficient viruses, in which the conserved CD4BS and CD4i epitopes are more exposed, better than the glycan-containing viruses. The titer of broadly neutralizing antibodies appears to be higher in the sera of macaques infected with glycan-deficient viruses. Collectively, our data add legitimacy to the use of SHIV-macaque models for testing the efficacy of HIV-1 Env-based immunogens. Furthermore, they suggest that antibodies to the CD4BS and CD4i sites of gp120 are prevalent in human and macaque sera and that the use of immunogens in which these conserved neutralizing epitopes are more exposed is likely to increase their immunogenicity.     

A high level of transgenic viral small RNA is associated with broad potyvirus resistance in cucurbits

Gene-silencing has been used to develop resistance against many plant viruses but little is known about the transgenic small-interfering RNA (t-siRNA) that confers this resistance. Transgenic cucumber and melon lines harboring a hairpin construct of the Zucchini yellow mosaic potyvirus (ZYMV) HC-Pro gene accumulated different levels of t-siRNA (6 to 44% of total siRNA) and exhibited resistance to systemic ZYMV infection. Resistance to Watermelon mosaic potyvirus and Papaya ring spot potyvirus-W was also observed in a cucumber line that accumulated high levels of t-siRNA (44% of total siRNA) and displayed significantly increased levels of RNA-dependent RNA (RDR)1 and Argonaute 1, as compared with the other transgenic and nontransformed plants. The majority of the t-siRNA sequences were 21 to 22 nucleotides in length and sense strand biased. The t-siRNA were not uniformly distributed throughout the transgene but concentrated in "hot spots" in a pattern resembling that of the viral siRNA peaks observed in ZYMV-infected cucumber and melon. Mutations in ZYMV at the loci associated with the siRNA peaks did not break this resistance, indicating that hot spot t-siRNA may not be essential for resistance. This study shows that resistance based on gene-silencing can be effective against related viruses and is probably correlated with t-siRNA accumulation and increased expression of RDR1.     

Yeast proteins associated with microtubules in vitro and in vivo.

Conditions were established for the self-assembly of milligram amounts of purified Saccharomyces cerevisiae tubulin. Microtubules assembled with pure yeast tubulin were not stabilized by taxol; hybrid microtubules containing substoichiometric amounts of bovine tubulin were stabilized. Yeast microtubule-associated proteins (MAPs) were identified on affinity matrices made from hybrid and all-bovine microtubules. About 25 yeast MAPs were isolated. The amino-terminal sequences of several of these were determined: three were known metabolic enzymes, two were GTP-binding proteins (including the product of the SAR1 gene), and three were novel proteins not found in sequence databases. Affinity-purified antisera were generated against synthetic peptides corresponding to two of the apparently novel proteins (38 and 50 kDa). Immunofluorescence microscopy showed that both these proteins colocalize with intra- and extranuclear microtubules in vivo.     

Enrichment and persistence of virus-specific CTL in the brain of simian immunodeficiency virus-infected monkeys is associated with a unique cytokine environment

The host reaction to infection of the brain contributes to a number of CNS pathologies including neuro-AIDS. In this study, we have identified the accumulation of SIV-specific CTL in the brains of SIV-infected animals who have neurophysiological abnormalities but are otherwise asymptomatic. SIV-specific CTL enter the brain early after viral infection and are maintained in the brain even when those reactive with an immunodominant epitope in Tat are lost from the rest of the body. The specialized CNS environment contributes to this unique outcome. Following SIV infection, brain levels of IL-15 were significantly elevated whereas IL-2 was absent, creating an environment that favors CTL persistence. Furthermore, in response to IL-15, brain-derived CD8(+) T cells could expand in greater numbers than those from spleen. The accumulation, persistence, and maintenance of CTL in the brain are closely linked to the increased levels of IL-15 in the absence of IL-2 in the CNS following SIV infection.     

Chemical changes associated with aging of collagen in vivo and in vitro

Collagen extracted from rat skin by neutral-salt solutions contains less aldehydes than the more insoluble collagen fractions. The concentration of aldehydes in collagen is directly related to its capacity to form stable cross-linked gels, which do not redissolve on cooling and become more insoluble in a variety of reagents. Whereas the absorption spectrum of neutral-salt-soluble collagen treated with N-methylbenzothiazolone hydrazone resembles that of acetaldehyde, the more insoluble collagen fractions show increasing amounts of a component that behaves like an alphabeta-unsaturated aldehyde. The ratio between alpha- and beta-sub-units present in a particular fraction of soluble collagen seems to be constant and independent of the age of the animal. Neutral-salt-soluble collagen, which has a low concentration of beta-components, will generate intramolecular bonds if gelled at 37 degrees . These intramolecular bonds seem to precede the formation of stable intermolecular cross-links, since these gels can redissolve when cooled to yield a soluble collagen with a higher content of beta-components of intramolecular origin.