Construction of combinatorial immune library of single chain human antibodies to orthopoxviruses and selection from this library antibodies to recombinant protein prA30L of variola virus

A combinatorial immune library of human single-chain antibody fragments (scFv) was constructed on the base of genes encoding variable domains of heavy and light chains of immunoglobulins cloned from the lymphocytes of four vaccinia virus (VACV) vaccinated donors. The size of the library was 3 x 10(7) independent clones. After the library was enriched with the clones producing scFv against recombinant analogue of variola virus surface protein prA30L, a panel of unique antibodies specific to both prA30L and VACV was selected from the library. A plaque reduction neutralization test was performed for all selected antibodies and two antibodies were shown to be able to neutralize plaque formation of VACV in Vero E6 cells monolayer. Binding specificities of these antibodies were confirmed using ELISA and Western blot analysis. To determine the amino acid sequences of neutralizing antibodies their genes were sequenced.     

Neutralizing Monoclonal Antibodies Cross-React with Fusion Proteins Encoded by <Emphasis Type="Italic">129L</Emphasis> of the Ectromelia Virus and <Emphasis Type="Italic">A30L</Emphasis> of the Variola Virus

PCR fragments containing the fusion protein genes 129L of the ectromelia virus (EV) and A30L of the variola virus (VARV) were cloned in pQE32. The expression products, recombinant prA30L and pr129L, were isolated from Escherichia coli cell lysates by metal-chelate affinity chromatography. The recombinant proteins retained the capability of oligomerization, characteristic of their natural analogs. ELISA and immunoblotting were used to test 22 monoclonal antibodies (mAbs) to orthopoxviruses (19 mAbs to EV, 2 mAbs to the vaccinia virus (VACV), and 1 mAb to the cowpox virus (CPXV)) for interaction with prA30L, pr129L, and orthopoxviruses. Twelve species-specific epitopes were found in the EV fusion protein 129L and its recombinant analog. Ten cross-reacting epitopes were found in the EV, CPXV, and VACV fusion proteins. Of these, nine epitopes were present both in prA30L and in the VARV fusion protein. Five mAbs interacting with cross-reacting epitopes were capable of efficient neutralization of VACV; two of these mAbs neutralized VARV. It was demonstrated that there are species-specific epitopes in EV 129L and cross-reacting epitopes in the EV, VARV, CPXV, and VACV fusion proteins, including epitopes that induced synthesis of virus-neutralizing antibodies against VACV and VARV.     

Fusion proteins encoded by orf 129L of ectromelia and orf A30L of smallpox viruses cross-react with neutralizing monoclonal antibodies

Open reading frame (orf) 129L of ectromelia (EV) and orf A30L of smallpox viruses (SPV) encoding fusion proteins were cloned and expressed in E. coli cells. The recombinant polypeptides (prA30L H pr129L) were purified from cell lysates by Ni-NTA chromatography. Recombinant polypeptides were able to form trimers in buffered saline and they destroyed under treatment with SDS and 2-mercaptoethanol. Reactivity of prA30L, pr129L and orthopoxvirus proteins was analyzed by ELISA and Western blotting with panel of 22 monoclonal antibodies (MAbs) against orthopoxviruses (19 against EV, 2 MAbs against vaccinia virus and 1 Mabs against cowpox virus). This data allowed us to conclude that there are 12 EV-specific epitopes of pr129L and EV fusion proteins, ten orthopox-specific epitopes of EV, VV, CPV fusion proteins, from them 9 orthopox-specific epitopes of prA30L and SPV fusion proteins. Five Mabs, which cross-reacted with orthopox-specific epitopes, were able to neutralize the VV on Vero cells and from them two MAbs has neutralizing activity against smallpox virus. Our findings demonstrate that 129L fusion protein have EV-specific epitopes, that EV 129L and SPV A30L fusion proteins have a several orthopox-specific epitopes to induce a neutralizing antibodies against human pathogenic orthopoxviruses.     

Role of sulfatide in vaccinia virus infection

Background information. Vaccinia virus (VACV) was used as a surrogate of variola virus (genus Orthopoxvirus), the causative agent of smallpox, to study orthopoxvirus infection. VACV infects cells via attachment and fusion of the viral membrane with the host cell membrane. Glycosphingolipids, expressed in multiple organs, are major components of lipid rafts and have been associated with the infectious route of several pathogens. Results. We demonstrate that the VACV‐WR (VACV Western‐Reserve strain) displays no binding to Cer (ceramide) or to Gal‐Cer (galactosylceramide), but binds to a natural sulfated derivative of these molecules: the Sulf (sulfatide) 3′ sulfogalactosylceramide. The interaction between Sulf and VACV‐WR resulted in a time‐dependent inhibition of virus infection. Virus cell attachment was the crucial step inhibited by Sulf. Electron microscopy showed that SUVs (small unilamellar vesicles) enriched in Sulf bound to VACV particles. Both the A27 and L5 viral membrane proteins were shown to interact with Sulf, indicating that they could be the major viral ligands for Sulf. Soluble Sulf was successful in preventing mortality, but not morbidity, in a lethal mouse model infection with VACV‐WR. Conclusions. Together the results suggest that Sulf could play a role as an alternate receptor for VACV‐WR and probably other Orthopoxviruses.     

C7L family of poxvirus host range genes inhibits antiviral activities induced by type I interferons and interferon regulatory factor 1

Vaccinia virus (VACV) K1L and C7L function equivalently in many mammalian cells to support VACV replication and antagonize antiviral activities induced by type I interferons (IFNs). While K1L is limited to orthopoxviruses, genes that are homologous to C7L are found in diverse mammalian poxviruses. In this study, we showed that the C7L homologues from sheeppox virus and swinepox virus could rescue the replication defect of a VACV mutant deleted of both K1L and C7L (vK1L(-)C7L(-)). Interestingly, the sheeppox virus C7L homologue could rescue the replication of vK1L(-)C7L(-) in human HeLa cells but not in murine 3T3 and LA-4 cells, in contrast to all other C7L homologues. Replacing amino acids 134 and 135 of the sheeppox virus C7L homologue, however, made it functional in the two murine cell lines, suggesting that these two residues are critical for antagonizing a putative host restriction factor which has some subtle sequence variation in human and murine cells. Furthermore, the C7L family of host range genes from diverse mammalian poxviruses were all capable of antagonizing type I IFN-induced antiviral activities against VACV. Screening of a library of more than 350 IFN-stimulated genes (ISGs) identified interferon-regulated factor 1 (IRF1) as an inhibitor of vK1L(-)C7L(-) but not wild-type VACV. Expression of either K1L or C7L, however, rendered vK1L(-)C7L(-) resistant to IRF1-induced antiviral activities. Altogether, our data show that K1L and C7L antagonize IRF1-induced antiviral activities and that the host modulation function of C7L is evolutionally conserved in all poxviruses that can readily replicate in tissue-cultured mammalian cells.     

牛痘病毒感染人巨噬细胞基因表达谱的RNA-Seq分析

[目的]利用RNA-Seq,分析人巨噬细胞在牛痘病毒(VACV)感染前后基因表达的变化,探索牛痘病毒与宿主细胞相互作用的机制.[方法]用牛痘病毒感染人巨噬细胞,采用RNA-Seq比较感染组和对照组的差异表达基因,并进行KEGG、GO以及STRING网络分析.[结果]感染组与对照组相比,筛选出显著性差异表达基因4796个...     

Recombinant full-size human antibody to Ebola virus

A full-size human antibody to Ebola virus was constructed by joining genes encoding the constant domains of the heavy and light chains of human immunoglobulin with the corresponding DNA fragments encoding variable domains of the single-chain antibody 4D1 specific to Ebola virus, which was chosen from a combinatorial phage display library of single-strand human antibodies. Two expression plasmids, pCH1 and pCL1, containing the artificial genes encoding the light and heavy chains of human immunoglobulin, respectively, were constructed. Their cotransfection into the human embryonic kidney cell line HEK293T provided the production of a full-size recombinant human antibody. The affinity constant for the antibody was estimated by solid-phase enzyme-linked immunoassay to be 7.7 × 10⁷ ± 1.5 × 10⁷ M^?1. Like the parent single-chain antibody 4D1, the resulting antibody bound the nucleoprotein of Ebola virus and did not interact with the proteins of Marburg virus.     

基于核糖体展示技术进行抗狂犬病毒人源抗体的制备

构建了核糖体展示人源抗狂犬病毒单链抗体(scFv)库,筛选制备特异抗狂犬病毒糖蛋白(RVGp)的稳定性人源抗体.应用核糖体抗体库技术,从经狂犬病毒Vero疫苗免疫的志愿者外周血淋巴细胞中分离、构建核糖体展示scFv基因库.体外转录翻译后,以RVGp重组蛋白作筛选抗原,采用亲和富集法淘选RVGp特异性scFv抗体基因.在原核系统pET22b(+)/BL21(DE3)中实现scFv抗体片段的可溶性表达,ELISA鉴定阳性克隆.然后对筛选的scFv进行稳定性改构,构建VH-Lc-VK稳定性抗体,并对其生物学活性进行初步研究.成功构建了库容量约为6.2×1012的核糖体展示scFv抗体基因库.在180个筛选克隆中,克隆RB24、RB71、RB109和RB156显示出较高的ELISA值,其基因序列分析结果显示,它们是全新的人源抗RVGp抗体.改构后的抗RVGp VH-Lc-VK抗体的稳定性明显改进,可特异识别RVGp并有效中和狂犬病毒,抑制狂犬病毒对靶细胞的感染.以上结果表明,人源抗RVGp特异性抗体的获得,为狂犬病的有效预防、诊断和治疗提供了新的途径,而且将为其他人源抗体的制备提供理论依据和技术基础.     

Genomic Sequence and Virulence of Clonal Isolates of Vaccinia Virus Tiantan,the Chinese Smallpox Vaccine Strain

Despite the worldwide eradication of smallpox in 1979, the potential bioterrorism threat from variola virus and the ongoing use of vaccinia virus (VACV) as a vector for vaccine development argue for continued research on VACV. In China, the VACV Tiantan strain (TT) was used in the smallpox eradication campaign. Its progeny strain is currently being used to develop a human immunodeficiency virus (HIV) vaccine. Here we sequenced the full genomes of five TT clones isolated by plaque purification from the TT (752-1) viral stock. Phylogenetic analysis with other commonly used VACV strains showed that TT (752-1) and its clones clustered and exhibited higher sequence diversity than that found in Dryvax clones. The ∼190 kbp genomes of TT appeared to encode 273 open reading frames (ORFs). ORFs located in the middle of the genome were more conserved than those located at the two termini, where many virulence and immunomodulation associated genes reside. Several patterns of nucleotide changes including point mutations, insertions and deletions were identified. The polymorphisms in seven virulence-associated proteins and six immunomodulation-related proteins were analyzed. We also investigated the neuro- and skin- virulence of TT clones in mice and rabbits, respectively. The TT clones exhibited significantly less virulence than the New York City Board of Health (NYCBH) strain, as evidenced by less extensive weight loss and morbidity in mice as well as produced smaller skin lesions and lower incidence of putrescence in rabbits. The complete genome sequences, ORF annotations, and phenotypic diversity yielded from this study aid our understanding of the Chinese historic TT strain and are useful for HIV vaccine projects employing TT as a vector.     

轮换淘选法筛选凝血酶特异的噬菌体抗体

以抗原为选择剂,通过淘选可以从噬菌体抗体库中获得特异的单链抗体克隆。采用液相一固相轮换淘选的方法,从鼠源噬菌体抗体库中淘选出与凝血酶特异结合的单链抗体。首先,用光敏生物素将凝血酶生物素化,然后用链亲和素磁珠法淘选与凝血酶特异结合的重组噬菌体。噬菌体扩增后使用酶标板进行第2轮淘选,以除去上一轮中非特异结合的重组噬菌体。经4轮轮换淘选,最后从23个单克隆噬菌体抗体中分离出4个凝血酶特异的噬菌体抗体。     

Improving Adaptive and Memory Immune Responses of an HIV/AIDS Vaccine Candidate MVA-B by Deletion of Vaccinia Virus Genes (C6L and K7R) Blocking Interferon Signaling Pathways

Poxvirus vector Modified Vaccinia Virus Ankara (MVA) expressing HIV-1 Env, Gag, Pol and Nef antigens from clade B (termed MVA-B) is a promising HIV/AIDS vaccine candidate, as confirmed from results obtained in a prophylactic phase I clinical trial in humans. To improve the immunogenicity elicited by MVA-B, we have generated and characterized the innate immune sensing and the in vivo immunogenicity profile of a vector with a double deletion in two vaccinia virus (VACV) genes (C6L and K7R) coding for inhibitors of interferon (IFN) signaling pathways. The innate immune signals elicited by MVA-B deletion mutants (MVA-B ΔC6L and MVA-B ΔC6L/K7R) in human macrophages and monocyte-derived dendritic cells (moDCs) showed an up-regulation of the expression of IFN-β, IFN-α/β-inducible genes, TNF-α, and other cytokines and chemokines. A DNA prime/MVA boost immunization protocol in mice revealed that these MVA-B deletion mutants were able to improve the magnitude and quality of HIV-1-specific CD4⁺ and CD8⁺ T cell adaptive and memory immune responses, which were mostly mediated by CD8⁺ T cells of an effector phenotype, with MVA-B ΔC6L/K7R being the most immunogenic virus recombinant. CD4⁺ T cell responses were mainly directed against Env, while GPN-specific CD8⁺ T cell responses were induced preferentially by the MVA-B deletion mutants. Furthermore, antibody levels to Env in the memory phase were slightly enhanced by the MVA-B deletion mutants compared to the parental MVA-B. These findings revealed that double deletion of VACV genes that act blocking intracellularly the IFN signaling pathway confers an immunological benefit, inducing innate immune responses and increases in the magnitude, quality and durability of the HIV-1-specific T cell immune responses. Our observations highlighted the immunomodulatory role of the VACV genes C6L and K7R, and that targeting common pathways, like IRF3/IFN-β signaling, could be a general strategy to improve the immunogenicity of poxvirus-based vaccine candidates.     

Identification and characterization of three immunodominant structural proteins of fowlpox virus

Genes encoding fowlpox virus (FWPV) structural proteins have been identified mainly by sequence homology with those from vaccinia virus (VACV), but little is known about the encoded proteins. Production of monoclonal antibodies (MAbs) against Poxine and HP1-440 (Munich) clone FP9 allowed the identification of three immunodominant FWPV proteins: the 39-kDa core protein (encoded by FPV168, homologous to VACV A4L), a 30- and 35-kDa protein doublet, and an abundant 63-kDa protein. The 30- and 35-kDa proteins are nonglycosylated, antigenically related proteins present in the intracellular mature virus membrane and localizing closely with the viral factories. N-terminal sequencing identified the 35-kDa protein as encoded by FPV140 (the FWPV homolog of VACV H3L). The 63-kDa protein forms covalently linked dimers and oligomers. It remained mainly insoluble upon detergent treatment of purified virus but did not localize closely with the viral factory. N-terminal sequencing was unsuccessful, suggesting N-terminal blocking. CNBr digestion generated a peptide encoded by FPV191, predicted to encode one of two FWPV A-type inclusion (ATI) proteins. The characteristics of the 63-kDa protein were inconsistent with published observations on cowpox or VACV ATI proteins (it appears to be essential). The 63-kDa protein, however, shares characteristics with both VACV p4c virus occlusion and 14-kDa fusion proteins. Gene assignment at the poxvirus ATI locus (between VACV A24R and A28L) is complicated by sequence redundancies and variations, often due to deletions and multiple frameshift mutations. The identity of FPV191 in relation to genes at this locus is discussed.     

Isolation and characterization of rice cesium transporter genes from a rice‐transporter‐enriched yeast expression library

A considerable portion of agricultural land in central‐east Japan has been contaminated by radioactive material, particularly radioactive Cs, due to the industrial accident at the Fukushima Daiichi nuclear power plant. Understanding the mechanism of absorption, translocation and accumulation of Cs⁺ in plants will greatly assist in developing approaches to help reduce the radioactive contamination of agricultural products. At present, however, little is known regarding the Cs⁺ transporters in rice. A transporter‐enriched yeast expression library was constructed and the library was screened for Cs⁺ transporter genes. The 1452 full length cDNAs encoding transporter genes were obtained from the Rice Genome Resource Center and 1358 clones of these transporter genes were successively subcloned into yeast expression vectors; which were then transferred into yeast. Using this library, both positive and negative selection screens can be performed, which have not been previously possible. The constructed library is an excellent tool for the isolation of novel transporter genes. This library was screened for clones that were sensitive to Cs⁺ using a SD‐Gal medium containing either 30 or 70 mM CsCl; resulting in the isolation of 13 Cs⁺ sensitive clones. ¹³⁷Cs absorption experiments were conducted and confirmed that all of the identified clones were able to absorb ¹³⁷Cs. A total of 3 potassium transporters, 2 ABC transporters and 1 NRAMP transporter were among the 13 identified clones.     

Vaccinia Virus K1L and C7L Inhibit Antiviral Activities Induced by Type I Interferons

Cellular tropism of vaccinia virus (VACV) is regulated by host range genes, including K1L, C7L, and E3L. While E3L is known to support viral replication by antagonizing interferon (IFN) effectors, including PKR, the exact functions of K1L and C7L are unclear. Here, we show that K1L and C7L can also inhibit antiviral effectors induced by type I IFN. In human Huh7 and MCF-7 cells, a VACV mutant lacking both K1L and C7L (vK1L⁻C7L⁻) replicated as efficiently as wild-type (WT) VACV, even in the presence of IFN. However, pretreating the cells with type I IFN, while having very little effect on WT VACV, blocked the replication of vK1L⁻C7L⁻ at the step of intermediate viral gene translation. Restoring either K1L or C7L to vK1L⁻C7L⁻ fully restored the IFN resistance phenotype. The deletion of K1L and C7L from VACV did not affect the ability of the virus to inhibit IFN signaling or its ability to inhibit the phosphorylation of PKR and the α subunit of eukaryotic initiation factor 2, indicating that K1L and C7L function by antagonizing an IFN effector(s) but with a mechanism that is different from those of IFN antagonists previously identified for VACV. Mutations of K1L that inactivate the host range function also rendered K1L unable to antagonize IFN, suggesting that K1L supports VACV replication in mammalian cells by antagonizing the same antiviral factor(s) that is induced by IFN in Huh7 cells.Vaccinia virus (VACV) is the prototypical member of the poxvirus family of large, complex, double-stranded DNA viruses (21). VACV has a very broad host range and is capable of infecting many vertebrate animal species. Its host range, however, can be significantly narrowed by deleting from its genome some of the so-called host range genes, the most important of which are E3L, K1L, and C7L (17). VACV mutants deleted of E3L (ΔE3L) or both K1L and C7L (ΔK1LΔC7L) replicate abortively and express only a subset of viral genes in most mammalian cell lines (3, 24). These mutants are highly attenuated in animal hosts but are capable of eliciting immune responses, making them attractive vaccine vectors for infectious diseases and cancers (27, 28). NYVAC, a VACV strain derived through deletion of 18 genes, including both K1L and C7L (27), has been used as the vector for an AIDS vaccine (2).The functions of E3L and the host factors that restrict the replication of the ΔE3L mutant have been studied extensively. E3L encodes a 20-kDa and a 25-kDa protein that bind double-stranded RNA (dsRNA) and Z form DNA (6, 15). The E3 proteins antagonize the dsRNA-dependent protein kinase PKR (5), which exists as an inactive form in the cells and undergoes autophosphorylation and activation upon binding to dsRNA. The activated PKR phosphorylates the α subunit of eukaryotic initiation factor 2 (eIF2α), resulting in a block in protein translation at the initiation step. The infection of most mammalian cells by the ΔE3L mutant leads to the activation of PKR and a block in translating viral mRNAs (16). The replication of the ΔE3L mutant in nonpermissive HeLa cells can be rescued by silencing PKR expression (32), while its replication in permissive Huh7 (human hepatoma) cells can be blocked by upregulating PKR expression with interferon (IFN) treatment (1). In addition to affecting PKR, E3 has also been shown to inactivate IFN-stimulated gene 15 (ISG15) (14), another IFN effector that plays a role in host defense against VACV.Like the replication of the ΔE3L mutant, the replication of the ΔK1LΔC7L mutant in nonpermissive HeLa cells is blocked at the translation of viral mRNA. However, the host factors that restrict the replication of the ΔK1LΔC7L mutant and the molecular functions of K1L or C7L remain a mystery. K1 and C7 share no amino acid sequence homology, but either K1L or C7L can complement the replication defect of the ΔK1LΔC7L mutant in most cell lines. The exception is rabbit RK13 cells, where K1L but not C7L can complement (24). K1L is present in only a few orthopoxviruses, while C7L or a functional homologue of C7L is present in almost all mammalian poxviruses (18). K1 comprises multiple ankyrin repeats, a protein motif that is involved in protein-ligand interaction. It was shown to prevent the degradation of IκBα and to thus inhibit host NF-κB activation in RK13 cells (25). C7L has no homologue outside the poxvirus family, and its molecular function remains unknown. It may play a role in inhibiting cellular apoptosis in response to VACV infection (23).As E3L supports VACV replication by antagonizing innate antiviral pathways that are inducible by IFNs, we hypothesize that K1L and C7L might function similarly by antagonizing IFN effectors. In the current study, we tested the hypothesis and found that both K1L and C7L can antagonize antiviral activities induced by type I IFNs. We found that K1L and C7L do not antagonize IFN by inhibiting IFN signaling or PKR activation, demonstrating that K1L and C7L are novel IFN antagonists functioning differently than previously identified IFN antagonists in VACV. Furthermore, we tested a panel of K1L mutant viruses and showed that K1L''s functions of regulating VACV host range and of antagonizing IFN are genetically nonsegregable, suggesting that K1L supports VACV replication in mammalian cells by antagonizing the same antiviral factor(s) that is induced by IFN in Huh7 cells.     

Anti-idiotypic antibodies induce neutralizing antibodies to rabies virus glycoprotein.

Rabbit anti-idiotypic antibodies (alpha Id Ab) were prepared against five murine monoclonal antibodies (mAb) specific for the rabies virus glycoprotein. Four of the mAb were directed against three known, type-specific, neutralizing sites on the glycoprotein, and the other mAb was directed against a topographically uncharacterized, nonneutralizing epitope. An absence of significant cross-reactivity among the alpha Id Ab for heterologous mAb suggested that the alpha Id Ab were highly specific for unique variable region determinants. The binding of three of the five alpha Id Ab to their homologous mAb could be inhibited by rabies virus-soluble glycoprotein, suggesting that the alpha Id Ab possessed subpopulations similar or adjacent to the antigen-binding site of the mAb. Two of the five alpha Id Ab injected into mice elicited a specific virus-neutralizing antibody response. Mechanisms to account for the induction of the virus-neutralizing antibody by alpha Id Ab are discussed.     

Improving the MVA vaccine potential by deleting the viral gene coding for the IL-18 binding protein

Background

Modified Vaccinia Ankara (MVA) is an attenuated strain of Vaccinia virus (VACV) currently employed in many clinical trials against HIV/AIDS and other diseases. MVA still retains genes involved in host immune response evasion, enabling its optimization by removing some of them. The aim of this study was to evaluate cellular immune responses (CIR) induced by an IL-18 binding protein gene (C12L) deleted vector (MVAΔC12L).

Methodology/Principal Findings

BALB/c and C57BL/6 mice were immunized with different doses of MVAΔC12L or MVA wild type (MVAwt), then CIR to VACV epitopes in immunogenic proteins were evaluated in spleen and draining lymph nodes at acute and memory phases (7 and 40 days post-immunization respectively). Compared with parental MVAwt, MVAΔC12L immunization induced a significant increase of two to three-fold in CD8⁺ and CD4⁺ T-cell responses to different VACV epitopes, with increased percentage of anti-VACV cytotoxic CD8⁺ T-cells (CD107a/b⁺) during the acute phase of the response. Importantly, the immunogenicity enhancement was also observed after MVAΔC12L inoculation with different viral doses and by distinct routes (systemic and mucosal). Potentiation of MVA''s CIR was also observed during the memory phase, in correlation with a higher protection against an intranasal challenge with VACV WR. Of note, we could also show a significant increase in the CIR against HIV antigens such as Env, Gag, Pol and Nef from different subtypes expressed from two recombinants of MVAΔC12L during heterologous DNA prime/MVA boost vaccination regimens.

Conclusions/Significance

This study demonstrates the relevance of IL-18 bp contribution in the immune response evasion during MVA infection. Our findings clearly show that the deletion of the viral IL-18 bp gene is an effective approach to increase MVA vaccine efficacy, as immunogenicity improvements were observed against vector antigens and more importantly to HIV antigens.     

Vaccinia virus H3L envelope protein is a major target of neutralizing antibodies in humans and elicits protection against lethal challenge in mice

The smallpox vaccine is the prototypic vaccine, yet the viral targets critical for vaccine-mediated protection remain unclear in humans. We have produced protein microarrays of a near-complete vaccinia proteome and used them to determine the major antigen specificities of the human humoral immune response to the smallpox vaccine (Dryvax). H3L, an intracellular mature virion envelope protein, was consistently recognized by high-titer antibodies in the majority of human donors, particularly after secondary immunization. We then focused on examining H3L as a valuable human antibody target. Purified human anti-H3L antibodies exhibited substantial vaccinia virus-neutralizing activity in vitro (50% plaque reduction neutralization test [PRNT50] = 44 microg/ml). Mice also make an immunodominant antibody response to H3L after vaccination with vaccinia virus, as determined by vaccinia virus protein microarray. Mice were immunized with recombinant H3L protein to examine H3L-specific antibody responses in greater detail. H3L-immunized mice developed high-titer vaccinia virus-neutralizing antibodies (mean PRNT50 = 1:3,760). Importantly, H3L-immunized mice were subsequently protected against lethal intranasal challenges with 1 or 5 50% lethal doses (LD50) of pathogenic vaccinia virus strain WR, demonstrating the in vivo value of an anti-H3L response. To formally demonstrate that neutralizing anti-H3L antibodies are protective in vivo, we performed anti-H3L serum passive-transfer experiments. Mice receiving H3L-neutralizing antiserum were protected from a lethal challenge with 3 LD50 of vaccinia virus strain WR (5/10 versus 0/10; P < 0.02). Together, these data show that H3L is a major target of the human anti-poxvirus antibody response and is likely to be a key contributor to protection against poxvirus infection and disease.     

Recombinant human monoclonal antibodies to human cytomegalovirus glycoprotein B neutralize virus in a complement-dependent manner

Human antibodies specific for HCMV are currently considered as potential anti-HCMV therapeutic agents. In this study, we used a combinatorial human antibody library to isolate and characterize complete human monoclonal antibodies that effectively neutralize HCMV in a complement-dependent manner. One hundred and six clones were isolated in two independent screens using HCMV virions and recombinant glycoprotein B, gB654, as antigens. All of the clones recognized the same molecule gB and were classified into 14 groups based on the amino acid sequence of the V_H region. Seven representative clones from these 14 groups had a strong gB654 binding affinity by surface plasmon resonance (SPR). A pairwise binding competition analysis suggested that there were three groups based on differences in the gB recognition sites. Although Fab fragments of the seven groups showed strong affinity for gB, none of the Fab fragments neutralized HCMV infectivity in vitro. In contrast, complete human IgG₁ antibodies of at least three groups neutralized HCMV in a complement-dependent manner. These data suggest that potent therapeutic antibodies can be obtained from a human antibody library, including most of the functional antibodies that mediate humoral immunity to the selected pathogen.