Colonic epithelium reactive monoclonal antibodies

Summary We have produced a small library of colonic mucosa and colorectal carcinoma reactive monoclonal antibodies (MoAbs) by immunizations with extracts of human colon cancer tissue and a human colon cancer cell line. Hybridoma supernatants were tested on (normal and neoplastic) human tissues by immunoperoxidase methods to evaluate organ or tissue specificity. Initial biochemical characterization of the target antigens was performed by gelpermeation chromatography, Western blotting and competition assays.Based upon the immunoreactivity patterns and the characteristics of the antigen four groups of MoAbs could be distinguished. The first group concerns the antibodies PAR-LAM 3, 9 and 10. These antibodies react with an 87 kDa protein moiety in high molecular weight (2–5×10⁶ Da) glycoproteins. In intestinal and colon mucosa these antibodies showed diffuse binding with goblet cells. In colon carcinoma decreased reactivity with these MoAbs was found.The second group consists of antibodies PARLAM 8, 12 and 13. These antibodies react with large (>5×10⁶ Da) glycoproteins, most likely with carbohydrate epitopes. By immunohistochemistry in normal colon mucosa the antibodies all show granular supranuclear reactivity with goblet cells. These antibodies show increased reactivity with colon adenomas and adenocarcinomas.A third group is formed by PARLAM 2, which also reacts with a large (>5×10⁶ Da) glycoprotein, showing a granular distribution in goblet cells. In colon carcinomas more extensive expression is found than in normal colonic mucosa. Finally, the fourth group consists of PARLAM 11, which also reacts with a large (>5×10⁶ Da) glycoprotein, located in the brush border of colonic columnar cells.These antibodies might be useful tools for the analysis of the expression of mucin related glycoproteins in normal, preneoplastic and neoplastic colon mucosa.Supported by grant RL 82-1 of the Netherlands Cancer Foundation, K.W.F.     

Design and analysis of rhesus cytomegalovirus IL-10 mutants as a model for novel vaccines against human cytomegalovirus

Background

Human cytomegalovirus (HCMV) expresses a viral ortholog (CMVIL-10) of human cellular interleukin-10 (cIL-10). Despite only ∼26% amino acid sequence identity, CMVIL-10 exhibits comparable immunosuppressive activity with cIL-10, attenuates HCMV antiviral immune responses, and contributes to lifelong persistence within infected hosts. The low sequence identity between CMVIL-10 and cIL-10 suggests vaccination with CMVIL-10 may generate antibodies that specifically neutralize CMVIL-10 biological activity, but not the cellular cytokine, cIL-10. However, immunization with functional CMVIL-10 might be detrimental to the host because of its immunosuppressive properties.

Methods and Findings

Structural biology was used to engineer biologically inactive mutants of CMVIL-10 that would, upon vaccination, elicit a potent immune response to the wild-type viral cytokine. To test the designed proteins, the mutations were incorporated into the rhesus cytomegalovirus (RhCMV) ortholog of CMVIL-10 (RhCMVIL-10) and used to vaccinate RhCMV-infected rhesus macaques. Immunization with the inactive RhCMVIL-10 mutants stimulated antibodies against wild-type RhCMVIL-10 that neutralized its biological activity, but did not cross-react with rhesus cellular IL-10.

Conclusion

This study demonstrates an immunization strategy to neutralize RhCMVIL-10 biological activity using non-functional RhCMVIL-10 antigens. The results provide the methodology for targeting CMVIL-10 in vaccine, and therapeutic strategies, to nullify HCMV''s ability to (1) skew innate and adaptive immunity, (2) disseminate from the site of primary mucosal infection, and (3) establish a lifelong persistent infection.     

A versatile bifunctional chelate for radiolabeling humanized anti-CEA antibody with In-111 and Cu-64 at either thiol or amino groups: PET imaging of CEA-positive tumors with whole antibodies

Radiolabeled anti-carcinoembryonic antigen (CEA) antibodies have the potential to give excellent images of a wide variety of human tumors, including tumors of the colon, breast, lung, and medullar thyroid. In order to realize the goals of routine and repetitive clinical imaging with anti-CEA antibodies, it is necessary that the antibodies have a high affinity for CEA, low cross reactivity and uptake in normal tissues, and low immunogenicity. The humanized anti-CEA antibody hT84.66-M5A (M5A) fulfills these criteria with an affinity constant of >10 (10) M (-1), no reactivity with CEA cross-reacting antigens found in normal tissues, and >90% human protein sequence. A further requirement for routine clinical use of radiolabeled antibodies is a versatile method of radiolabeling that allows the use of multiple radionuclides that differ in their radioemissions and half-lives. We describe a versatile bifunctional chelator, DO3A-VS (1,4,7-tris(carboxymethyl)-10-(vinylsulfone)-1,4,7,10-tetraazacyclododecane) that binds a range of radiometals including 111 In for gamma-ray imaging and 64Cu for positron emission tomography (PET), and which can be conjugated with negligible loss of immunoreactivity either to sulfhydryls (SH) in the hinge region of lightly reduced immunoglobulins or surface lysines (NH) of immunoglobulins. Based on our correlative studies comparing the kinetics of radiolabeled anti-CEA antibodies in murine models with those in man, we predict that 64Cu-labeled intact, humanized antibodies can be used to image CEA positive tumors in the clinic.     

长角血蜱卵黄蛋白单克隆抗体的制备及鉴定

为研究蜱类的卵黄发生及其机理,用长角血蜱Haemaphysalis longicornis卵黄蛋白（vitellin, Vn）免疫BALB/c小鼠,取免疫鼠脾细胞与骨髓瘤细胞SP2/0进行融合,经3次克隆化筛选,获得6株能稳定分泌抗Vn的单克隆抗体（McAb）,即1B5,2A7,2B8,2F2,3A1和3G1。1B5,2B8和2F2亚型为IgGA;2A7亚型为IgG1;3A1和3G1亚型为IgG2a。6株McAb均具有高度特异性,效价在1∶10.5以上。选取效价和特异性最好的1株抗体1B5进行SDS-PAGE分析和亲和力测定,测得1B5重链和轻链的分子量分别为58 kD和21 kD,亲和常数为2.8993×10^-6。Western免疫印迹分析发现6株单抗均与Vn的8个亚基发生免疫反应。本研究成功制备了6株抗长角血蜱Vn的单克隆抗体,为深入阐明长角血蜱卵黄发生的过程与调控机理提供了重要的工具。     

Protective effects of anti-C5a peptide antibodies in experimental sepsis.

We evaluated antibodies to different peptide regions of rat C5a in the sepsis model of cecal ligation and puncture (CLP) for their protective effects in rats. Rabbit polyclonal antibodies were developed to the following peptide regions of rat C5a: amino-terminal region (A), residues 1-16; middle region (M), residues 17-36; and the carboxyl-terminal region (C), residues 58-77. With rat neutrophils, the chemotactic activity of rat C5a was significantly inhibited by antibodies with the following rank order: anti-C > anti-M > anti-A. In vivo, antibodies to the M and C (but not A) regions of C5a were protective in experimental sepsis, as determined by survival over a 10-day period, in a dose-dependent manner. The relative protective efficacies of anti-C5a preparations (in descending order of efficacy) were anti-C > anti-M > anti-A. In CLP rats, a delay in infusion of antibodies, which were injected at 6 or 12 h after CLP, still resulted in significant improvement in survival rates. These in vivo and in vitro data suggest that there are optimal targets on C5a for blockade during sepsis and that delayed infusion of anti-C5a antibody until after onset of clinical evidence of sepsis still provides protective effects.     

Human Immunodeficiency Virus Type-1 (HIV-1) Continues to Evolve in Presence of Broadly Neutralizing Antibodies More than Ten Years after Infection

Background

The evolution of HIV-1 and its immune escape to autologous neutralizing antibodies (Nabs) during the acute/early phases of infection have been analyzed in depth in many studies. In contrast, little is known about neither the long-term evolution of the virus in patients who developed broadly Nabs (bNabs) or the mechanism of escape in presence of these bNabs.

Results

We have studied the viral population infecting a long term non progressor HIV-1 infected patient who had developed broadly neutralizing antibodies toward all tier 2/3 viruses (6 clades) tested, 9 years after infection, and was then followed up over 7 years. The autologous neutralization titers of the sequential sera toward env variants representative of the viral population significantly increased during the follow-up period. The most resistant pseudotyped virus was identified at the last visit suggesting that it represented a late emerging escape variant. We identified 5 amino acids substitutions that appeared associated with escape to broadly neutralizing antibodies. They were V319I/S, R/K355T, R/W429G, Q460E and G/T463E, in V3, C3 and V5 regions.

Conclusion

This study showed that HIV-1 may continue to evolve in presence of both broadly neutralizing antibodies and increasing autologous neutralizing activity more than 10 years post-infection.     

1H NMR study of the binding of bis(acridines) to d(AT)5.d(AT)5. 2. Dynamic aspects

Measurements of the 1H NMR spectra and relaxation rates were used to study the dynamic properties of 9-aminoacridine (9AA) and four bis(acridine) complexes with d(AT)5.d(AT)5. The behavior of the 9AA (monointercalator) and that of C8 (bisintercalator containing an eight-carbon atom linker chain) are entirely similar. For both compounds, the lifetime of the drug in a particular binding site is 2-3 ms at approximately 20 degrees C, and neither affects the A.T base pair opening rates. The complex with C10 (bisintercalator containing a 10-carbon atom linker chain) is slightly more stable than the C8 complex since its estimated binding site lifetime is 5-10 ms at 29 degrees C. Base pairs adjacent to the bound C10 are destabilized, relative to free d(AT)5.d(AT)5, but other base pairs in the C10 complex are little affected. Bis(acridine) pyrazole (BAPY) and bis(acridine) spermine (BAS) considerably stabilize those base pairs that are sandwiched between the two acridine chromophores, but in the BAS complex proton exchange from the two flanking base pairs appears to be accelerated, relative to free d(AT)5.d(AT)5. The lifetime of these drugs in specific binding sites is too long (>10 ms) to be manifested in increased line widths, at least up to 41 degrees C. An important conclusion from this study is that certain bisintercalators rapidly migrate along DNA, despite having large binding constants (K>10(6) M-1). For C8 and C10 complexes, migration rates are little different from those deduced for 9AA. The rigid linker chain in BAPY and the charge interactions in BAS retard migration of these two bisintercalators. These results provide new parameters that are useful in understanding the biochemical and biological properties of these and other bisintercalating drugs.     

Base specificity and idiotypy of anti-DNA autoantibodies reactive with synthetic nucleic acids

Synthetic nucleic acid reactivities and the distribution of idiotypes associated with poly(dA) and poly(dT) specificities were evaluated among both monoclonal and polyclonal anti-DNA antibodies from autoimmune New Zealand mice. Ten monoclonal anti-DNA antibodies (IgG2a or IgG2b), derived from NZB/NZW mice and reactive with natural DNA (duplex and/or heat-denatured), were found to collectively exhibit a diverse binding pattern with six deoxyribohomopolymers. Several monoclonal antibodies displayed reactivity with poly(dT) comparable to that with natural DNA. Serologic studies indicated that polyclonal anti-DNA autoantibodies from NZW/NZW mice and both parental strains also cross-reacted with various homopolymers and bound preferentially with those containing pyrimidines, particularly poly(dT), relative to purines. Detailed binding analyses with two poly(dT)-reactive monoclonal antibodies demonstrated that stable DNA/anti-DNA complexes were formed with synthetic oligomers containing six to 10 nucleotides; binding to such antigens was relatively insensitive to ionic strength and inversely dependent on temperature. Both antibodies exhibited preferential binding (greater than or equal to 10-fold) with poly(dT) relative to poly(dU), suggesting the importance of the C5-methyl group and/or helical conformation in pyrimidine base recognition. Idiotypes on poly(dA)-specific and poly(dT)-specific monoclonal antibodies were found to be reciprocally distinct, localized at or near active site residues, and expressed at low levels (less than 10 to 130 ng/ml) in anti-DNA sera from all three New Zealand strains. These findings suggest that: nucleotide base determinants are significantly involved in DNA/anti-DNA interactions; poly(dT) represents a major cross-reactive synthetic antigen; and idiotype expression among lupus autoantibodies which recognize such determinants may be diverse.     

Two induced anti-Z-DNA monoclonal antibodies use VH gene segments related to those of anti-DNA autoantibodies

The cDNA for H and L chain V regions of two anti-Z-DNA mAb, Z22 and Z44, were cloned and sequenced. These are the first experimentally induced anti-nucleic acid antibody sequences available for comparison with autoantibody sequences. Z22 and Z44 are IgG2b and IgG2a antibodies from C57BL/6 mice. They recognize different facets of the Z-DNA structure. They both use VH10 family genes and share 95% sequence base sequence identity in the VH and leader sequences; however, they differ in the 5'-untranslated region of the VH mRNA, indicating they arise from different germline genes. Both use JH4 segments. They differ from each other very extensively in the CDR3 of both H and L chains. The most closely related H chains in the current GenBank/EMBL data base are two mouse IgG anti-DNA autoantibodies, one from an MRL-lpr/lpr mouse (MRL-DNA4) and one from an NZB/NZW mouse (BV04-01). Z22 and Z44 share 95% sequence identity with these antibodies in the VH segment. In addition, Z22 is identical to MRL-DNA4 at 91% of the positions in the 5'-untranslated region of the H chain mRNA. The two antibodies share 95% base sequence identity in the V kappa segment. The most closely related L chains, with 97 to 98% sequence identity, are the V kappa 10b germline gene for Z22 and the V kappa 10a germ line gene, which is associated with A/J anti-arsonate antibodies and BALB/c anti-ABO blood group substance antibodies, for Z44. Z22 and Z44 share several structural features (similarities in VH, JH, and V kappa) but differ very markedly in the L chain CDR1 and both H and L chain CDR3 sequences; these regions may determine the differences in their specific interactions with Z-DNA.     

In vitro toxicity of palladium(II) and gold(III) porphyrins and their aqueous metal ion counterparts on Trypanosoma brucei brucei growth

The trypanocidal effects of aqueous gold(III) and palladium(II) and their metalloporphyrin derivatives on Trypanosoma brucei brucei growth in culture have been studied using an Alamar Blue indicator assay. All the experiments were conducted in the dark. As previously described for mercury(II), cadmium(II) and lead(II) porphyrins [Chem.-Biol. Interact. 139 (2002) 177], the toxicity of the metalloporphyrin complex of palladium(II) to T. b. brucei parasites was much higher compared to the aqueous free palladium(II) and free base porphyrin. Palladium(II) porphyrin, free palladium(II), and the free base porphyrin were trypanocidal to T. b. brucei at concentrations >1.5 x 10(-6), >6.1 x 10(-6) and >1.9 x 10(-5) M, respectively. While gold(III) porphyrin was effective against the parasites at concentrations >4.8 x 10(-6) M, its aqueous gold(III) was toxic at concentrations as low as 2.0 x 10(-7) M due to the generation of free radicals in the presence of this metal ion which enhanced its toxicity to the T. b. brucei parasites. Although some cell division was observed in some of the cells treated with palladium(II) porphyrin, some dividing cells had no nucleus due to unequal division and delivery of the nuclei into the daughter cells. As a result, the rate of cell division decreased with time and cell death occurred within 24 h. Interestingly, trypanosomes treated with metalloporphyrin complexes displayed different morphological features from those cells treated with free base porphyrin or metal ions. Of all the porphyrins and free metal ions tested, only mercury(II) porphyrin and aqueous gold(III) ion were toxic to the trypanosomes in the 10(-7) M range. The chemotherapeutic potential of these observations is discussed.     

A label-free continuous total-internal-reflection-fluorescence-based immunosensor

In this study, we continuously monitored, second-by-second, concentration changes of two different carbohydrates (maltose and panose) by using monoclonal antibodies in an optical immunosensor based on total internal reflection fluorescence. Earlier studies have demonstrated that these antibodies increase their intrinsic tryptophan fluorescence upon binding of carbohydrate antigens. Using the four immobilized monoclonal antibodies with low affinities (K(d)>10(-6)M), fast kinetics (k(off)>1s(-1)), and high reversibility gave opportunities for developing a continuous immunosensor without any need for regeneration. Since intrinsic fluorescence was used, no extrinsic labeling was necessary. Sensitivity was in the range of 1-5 microM for panose, and 10-15 microM for maltose and the loss of intensity was as low as 3.5% per hour during measurements. Calculations of DeltaH degrees and DeltaS degrees from the temperature dependence of K(d) indicated an enthalpic driven antigen-antibody binding event that is diminished upon antibody immobilization. We feel certain that weakly interacting antibodies can be used in future applications for continuous monitoring where there is a need to achieve instantaneous information on the concentration of an analyte.     

Formation of modified DNA bases in cells exposed either to gamma radiation or to high-LET particles

The aim of the present study was to measure the formation of eight base modifications in the DNA of cells exposed to either low-LET ((60)Co gamma rays) or high-LET ((12)C(6+) particles) radiation. For this purpose, a recently optimized HPLC-MS/MS method was used subsequent to DNA extraction and hydrolysis. The background level of the measured modified bases and nucleosides was shown to vary between 0.2 and 2 lesions/10(6) bases. Interestingly, thymidine glycols constitute the main radiation-induced base modifications, with an overall yield of 0.097 and 0.062 lesion/10(6) bases per gray for gamma rays and carbon heavy ions, respectively. Both types of radiations generate four other major degradation products, in the following order of decreasing importance: FapyGua > 5-HmdUrd > 5-FordUrd > 8-oxodGuo. The yields of formation of FapyAde and 8-oxoAde are one order of magnitude lower than those of the related guanine modifications, whereas the radiation-induced generation of 5-OHdUrd was below the limit of detection of the assay. The efficiency for both types of radiation to generate base damage in cellular DNA is low because the highest yield per gray was 0.097 thymine glycols per 10(6) DNA bases. As a striking observation, the yield of formation of the measured DNA lesions was found to be, on average, twofold lower after exposure to high-LET radiation ((12)C(6+)) than after exposure to low-LET gamma radiation. These studies show that the HPLC-MS/MS assay provides an accurate, reliable and sensitive method for measuring cellular DNA base damage.     

Potent neutralization of botulinum neurotoxin/b by synergistic action of antibodies recognizing protein and ganglioside receptor binding domain

Background

Botulinum neurotoxins (BoNTs), the causative agents for life-threatening human disease botulism, have been recognized as biological warfare agents. Monoclonal antibody (mAb) therapeutics hold considerable promise as BoNT therapeutics, but the potencies of mAbs against BoNTs are usually less than that of polyclonal antibodies (or oligoclonal antibodies). The confirmation of key epitopes with development of effective mAb is urgently needed.

Methods and Findings

We selected 3 neutralizing mAbs which recognize different non-overlapping epitopes of BoNT/B from a panel of neutralizing antibodies against BoNT/B. By comparing the neutralizing effects among different combination groups, we found that 8E10, response to ganglioside receptor binding site, could synergy with 5G10 and 2F4, recognizing non-overlapping epitopes within Syt II binding sites. However, the combination of 5G10 with 2F4 blocking protein receptor binding sites did not achieve synergistical effects. Moreover, we found that the binding epitope of 8E10 was conserved among BoNT A, B, E, and F, which might cross-protect the challenge of different serotypes of BoNTs in vivo.

Conclusions

The combination of two mAbs recognizing different receptors'' binding domain in BoNTs has a synergistic effect. 8E10 is a potential universal partner for the synergistical combination with other mAb against protein receptor binding domain in BoNTs of other serotypes.     

Potent and broad neutralizing activity of a single chain antibody fragment against cell-free and cell-associated HIV-1

Several human monoclonal antibodies (hmAbs) exhibit relatively potent and broad neutralizing activity against HIV-1, but there has not been much success in using them as potential therapeutics. We have previously hypothesized and demonstrated that small engineered antibodies can target highly conserved epitopes that are not accessible by full-size antibodies. However, their potency has not been comparatively evaluated with known HIV-1-neutralizing hmAbs against large panels of primary isolates. We report here the inhibitory activity of an engineered single chain antibody fragment (scFv), m9, against several panels of primary HIV-1 isolates from group M (clades A–G) using cell-free and cell-associated virus in cell line-based assays. M9 was much more potent than scFv 17b, and more potent than or comparable to the best-characterized broadly neutralizing hmAbs IgG₁ b12, 2G12, 2F5 and 4e10. It also inhibited cell-to-cell transmission of HIV-1 with higher potency than enfuvirtide (t-20, Fuzeon). M9 competed with a sulfated CCR5 N-terminal peptide for binding to gp120-CD4 complex, suggesting an overlapping epitope with the coreceptor binding site. M9 did not react with phosphatidylserine (pS) and cardiolipin (CL), nor did it react with a panel of autoantigens in an antinuclear autoantibody (ANA) assay. We further found that escape mutants resistant to m9 did not emerge in an immune selection assay. these results suggest that m9 is a novel anti-HIV-1 candidate with potential therapeutic or prophylactic properties, and its epitope is a new target for drug or vaccine development.Key words: HIV, AIDS, antibodies, scFv, microbicides, therapeutics, vaccines     

Genetic correction of splice site mutation in purified and enriched myoblasts isolated from mdx5cv mice

Background  

Duchenne Muscular Dystrophy (DMD) is an X-linked genetic disorder that results in the production of a dysfunctional form of the protein, dystrophin. The mdx^5cv mouse is a model of DMD in which a point mutation in exon 10 of the dystrophin gene creates an artificial splice site. As a result, a 53 base pair deletion of exon 10 occurs with a coincident creation of a frameshift and a premature stop codon. Using primary myoblasts from mdx^5cv mice, single-stranded DNA oligonucleotides were designed to correct this DNA mutation.     

Differential Specificity and Immunogenicity of Adenovirus Type 5 Neutralizing Antibodies Elicited by Natural Infection or Immunization

A recent clinical trial of a T-cell-based AIDS vaccine delivered with recombinant adenovirus type 5 (rAd5) vectors showed no efficacy in lowering viral load and was associated with increased risk of human immunodeficiency virus type 1 (HIV-1) infection. Preexisting immunity to Ad5 in humans could therefore affect both immunogenicity and vaccine efficacy. We hypothesized that vaccine-induced immunity is differentially affected, depending on whether subjects were exposed to Ad5 by natural infection or by vaccination. Serum samples from vaccine trial subjects receiving a DNA/rAd5 AIDS vaccine with or without prior immunity to Ad5 were examined for the specificity of their Ad5 neutralizing antibodies and their effect on HIV-1 immune responses. Here, we report that rAd5 neutralizing antibodies were directed to different components of the virion, depending on whether they were elicited by natural infection or vaccination in HIV vaccine trial subjects. Neutralizing antibodies elicited by natural infection were directed largely to the Ad5 fiber, while exposure to rAd5 through vaccination elicited antibodies primarily to capsid proteins other than fiber. Notably, preexisting immunity to Ad5 fiber from natural infection significantly reduced the CD4 and CD8 cell responses to HIV Gag after DNA/rAd5 vaccination. The specificity of Ad5 neutralizing antibodies therefore differs depending on the route of exposure, and natural Ad5 infection compromises Ad5 vaccine-induced immunity to weak immunogens, such as HIV-1 Gag. These results have implications for future AIDS vaccine trials and the design of next-generation gene-based vaccine vectors.Recombinant adenovirus (rAd)-based vectors are currently under investigation in a variety of gene therapy and T-cell-based vaccine clinical trials. There are more than 370 such ongoing clinical trials for broad applications, including infectious diseases and cancer therapy (http://www.wiley.co.uk/genetherapy/clinical/). Based on supportive data from nonhuman primate studies, rAd-based vectors have been developed and tested in human clinical trials to deliver human immunodeficiency virus (HIV-1) gene products that stimulate HIV-specific immune responses. Preexisting immunity to Ad serotype 5 (Ad5), from which most vectors are derived, is common in humans. Though neutralizing antibodies to Ad5 may reduce the immunogenicity of Ad5-based vectors in animal models (16), their effect on immunity in subjects with previous Ad5 infection is poorly understood. In the STEP trial, which tested a Merck rAd5 vaccine encoding HIV-1 Gag, Pol, and Nef, vaccination failed to show protection, either by lowering viral load or by decreasing acquisition of infection (3, 9, 12, 21). Furthermore, the possibility was raised that subjects with preexisting neutralizing antibodies from natural Ad5 infection may have carried an increased risk of HIV infection after vaccination. Thus, understanding the nature and immune effects of Ad5 seropositivity in humans is important to the development of vaccines against AIDS and other diseases.Ad5 is a common cause of respiratory disease and an occasional cause of gastroenteritis in humans, and exposure before adolescence is common in human populations (19). Such exposure stimulates both innate and adaptive immune responses that generate neutralizing antibodies and virus-specific T-cell responses (6). These antibodies can also synergize with each other to achieve maximum viral neutralization (7, 22). The capsid protein specificity of Ad5 neutralizing antibodies has been reported for humans following administration of rAd5 gene therapy vectors for advanced liver or lung cancer (7, 10). However, results were presented solely for antibodies induced by administration of rAd5. One report has assessed Ad5 neutralizing antibodies with a healthy human population that was Ad5 seropositive from natural exposure to the virus (18). The median titer of the population was presented, but the frequency of protein-specific neutralizing antibody has not been defined for humans.Here we describe the first report of the natural frequency and effect on immunization of neutralizing antibodies specific for different Ad capsid proteins in human subjects. We address the fundamental mechanisms of how humans generate neutralizing antibodies to a common cold virus that is in widespread use as a vector for gene therapy and vaccines. Such mechanisms may also be applicable to other nonenveloped viruses, including adeno-associated viruses and other viruses containing multiple envelope surface proteins, like influenza. To analyze the contribution of anti-capsid antibodies to neutralization by different human serum samples, wild-type and chimeric vectors were utilized. For example, a rAd type 5 (rAd5) vector with a fiber derived from Ad35 fiber (rAd5 F35) can be used to analyze the anti-Ad5 capsid response independent of fiber. Conversely, a rAd35 vector with a fiber transposed from Ad5 can determine the specificity of neutralization mediated by the Ad5 fiber. Using these vectors, we have analyzed human serum samples from two HIV vaccine clinical trials, VRC 006 and HVTN 204, in which a single-dose rAd5 vaccine alone and a three-dose DNA prime/single dose rAd5 boost vaccine encoding HIV-1 Env A,B, and C; Gag; and Pol, respectively, were administered. Thus, we sought to characterize the specificity of rAd5 neutralizing antibodies in Ad5-immune subjects and to determine their effect on immune responses elicited by vaccination.     

Characterization of a Large Panel of Rabbit Monoclonal Antibodies against HIV-1 gp120 and Isolation of Novel Neutralizing Antibodies against the V3 Loop

We recently reported the induction of potent, cross-clade neutralizing antibodies (nAbs) against Human Immunodeficiency Virus type-1 (HIV-1) in rabbits using gp120 based on an M-group consensus sequence. To better characterize these antibodies, 93 hybridomas were generated, which represent the largest panel of monoclonal antibodies (mAbs) ever generated from a vaccinated rabbit. The single most frequently recognized epitope of the isolated mAbs was at the very C-terminal end of the protein (APTKAKRRVVEREKR), followed by the V3 loop. A total of seven anti-V3 loop mAbs were isolated, two of which (10A3 and 10A37) exhibited neutralizing activity. In contrast to 10A3 and most other anti-V3 loop nAbs, 10A37 was atypical with its epitope positioned more towards the C-terminal half of the loop. To our knowledge, 10A37 is the most potent and broadly neutralizing anti-V3 loop mAb induced by vaccination. Interestingly, all seven anti-V3 loop mAbs competed with PGT121, suggesting a possibility that early induction of potent anti-V3 loop antibodies could prevent induction of more broadly neutralizing PGT121-like antibodies that target the conserved base of the V3 loop stem.     

Toxic effects of mercury(II), cadmium(II) and lead(II) porphyrins on Trypanosoma brucei brucei growth

The effects of free mercury(II), cadmium(II) and lead(II) ions and their metalloporphyrin-derivatives on Trypanosoma brucei brucei growth in culture were studied. All experiments were conducted in the dark. IC(50) values on growth obtained in 24-h time-course experiments were 1.5 x 10(-7), 2.4 x 10(-6), 4.4 x 10(-6) and 2.6 x 10(-5) M for mercury(II) porphyrin, cadmium(II) porphyrin, lead(II) porphyrin and free base porphyrin, respectively. While the IC50 values for Hg2+, Cd2+ and Pb2+ were 3.6 x 10(-6), 1.5 x 10(-5) and 1.6 x 10(-5) M, respectively. These results clearly indicate that the toxicity of the metalloporphyrin complexes of mercury(II), cadmium(II) and lead(II) to T. b. brucei parasites was much higher compared to their free metal ions and free base porphyrin at low concentrations. It was also observed after 8 h incubation that the metalloporphyrins were effective in inhibiting the division of the parasites at concentrations >1.25 x 10(-7) M for mercury(II) porphyrin, concentrations >1.2 x 10(-6) M for cadmium(II) and lead(II) porphyrins and at concentrations >3.6 x 10(-6) M for Hg2+ ion. These observations were not detected in samples treated with the free metal ions and the free base porphyrin at the same concentrations. Interestingly, trypanosomes treated with metalloporphyrin complexes displayed different morphological features from those cells treated with free base porphyrin or metal ions. The chemotherapeutic potential of the metalloporphyrins of H2TMPyP for treatment of African trypanosomiasis is discussed.     

Development and immunological assessment of VLP-based immunogens exposing the membrane-proximal region of the HIV-1 gp41 protein

Background

The membrane-proximal external region (MPER) of HIV-1 gp41 is particularly conserved and target for the potent broadly neutralizing monoclonal antibodies (bnMAbs) 2F5, 4E10 and 10E8. Epitope focusing and stabilization present promising strategies to enhance the quality of immune responses to specific epitopes.

Results

The aim of this work was to design and evaluate novel immunogens based on the gp41 MPER with the potential to elicit cross-clade neutralizing antibodies. For that purpose, gp41 was truncated N-terminally in order to dispose immunodominant, non-neutralizing sites and enhance the exposure of conserved regions. To stabilize a trimeric conformation, heterologous GCN4 and HA2 zipper domains were fused based on an in silico “best-fit” model to the protein’s amino terminus. Cell surface exposure of resulting proteins and their selective binding to bnMAbs 2F5 and 4E10 could be shown by cytometric analyses. Incorporation into VLPs and preservation of antigenic structures were verified by electron microscopy, and the oligomeric state was successfully stabilized by zipper domains. These gp41 immunogens were evaluated for antigenicity in an immunization study in rabbits primed with homologous DNA expression plasmids and boosted with virus-like particle (VLP) proteins. Low titers of anti-MPER antibodies were measured by IgG ELISA, and low neutralizing activity could be detected against a clade C and B viral isolate in sera.

Conclusions

Thus, although neutralizing titers were very moderate, induction of cross-clade neutralizing antibodies seems possible following immunization with MPER-focusing immunogens. However, further refinement of MPER presentation and immunogenicity is clearly needed to induce substantial neutralization responses to these epitopes.

Electronic supplementary material

The online version of this article (doi:10.1186/s12929-014-0079-x) contains supplementary material, which is available to authorized users.