Antibody VH domain sequence analysis by a bioinformatics approach based on electronic amino acid properties may help to predict paratop location.

Gene as the basic functional unit of DNA encodes information about the product such as protein. The majority of proteins realize function through protein-protein interactions involving short protein motifs. However, some proteins such as antibodies are established by the rearrangement of several (V-D-J) gene segments with the potential addition of nontemplated nucleotides that may change information encoded by the respective gene segment used. Antibody VH domain sequence analysis by ISM bioinformatics approach that is based on amino acids physicochemical features, enable to distinguish the contribution of the information encoded by VH gene or generated during VDJ gene recombination for antibody-antigen interaction. The data presented in this report revealed the significance of CDRH3 for the interaction of antibody specific for immunogenic molecules while CDRH3 contribution is minor for antibody interaction with nonimmunogenic molecules such as haptens and native mammalian dsDNA. Thus, paratopes might be located in the CDRH3 or VH regions.

A Model for Potential B-cell Precursors of Broadly Neutralizing HIV-1 Antibodies Selection and Antibody Affinity Maturation

The goal of this report was to propose a model, wherein synergy between the B-cell antigen receptor (BCR) and toll-like receptor (TLR) signaling is involved in the selection of the B-cell precursors of HIV-1 broadly neutralizing antibodies (bnAbs) with long heavy chain complementarity determining regions 3, from immature/transitional B cells. The model predicts the involvement of Ab/HIV-1 complexes in a way that Ab from the complex binds both BCRs and HIV-1, while on internalization of HIV-1 TLR ligands such as CpG motifs interacts with TLR9. The result of BCR and TLR9 orchestrated signaling is a formation of somatically mutated memory B cells potential precursors of bnAbs. Generated memory B cells continuously exposed to different Ab/HIV-1 complexes can elicit specific bnAb by stochastic somatic hypermutation rather than in the Darwinian process. This new view of the interaction between Ab/HIV-1 complexes and immune system, leading to affinity maturation of the bnAbs in the absence of nominal HIV-1 antigen and BCR interaction, may have implication for the vaccine designed and passive immunization.

Antibody Epitope Specificity for dsDNA Phosphate Backbone Is an Intrinsic Property of the Heavy Chain Variable Germline Gene Segment Used.

Analysis of protein sequences by the informational spectrum method (ISM) enables characterization of their specificity according to encoded information represented with defined frequency (F). Our previous data showed that F(0.367) is characteristic for variable heavy chain (VH) domains (a combination of variable (V), diversity (D) and joining (J) gene segments) of the anti-phosphocholine (PC) T15 antibodies and mostly dependent on the CDR2 region, a site for PC phosphate group binding. Because the T15 dsDNA-reactive U4 mutant also encodes F(0.367), we hypothesized that the same frequency may also be characteristic for anti-DNA antibodies. Data obtained from an analysis of 60 spontaneously produced anti-DNA antibody VH domain sequences supported our hypothesis only for antibodies, which use V gene segment in germline configuration, such as S57(VH31), MRL-DNA22, and VH11, members of the VH1 (J558) and VH7 (S107) gene families. The important finding is that out of seven V gene segments used by spontaneous anti-DNA antibodies, F(0.367) is only expressed by the germline configuration of these three V gene segments. The data suggest that antibody specificity for the phosphate group moiety delineated as F(0.367) is the intrinsic property of the V germline gene segments used, whereas paratope/epitope interaction with antigens bearing this epitope, such as PC or dsDNA, requires corresponding antibody VH conformation that is susceptible to somatic mutation(s).

Phosphocholine-binding antibody activities are hierarchically encoded in the sequence of the heavy-chain variable region: dominance of self-association activity in the T15 idiotype.

A methodology based on the representation of each amino acid of a protein sequence by the electron-ion interaction potential and subsequent analysis by signal processing was used to determine the characteristic or common frequency (in Hz) that reflects the biological activity shared among phosphocholine (PC)-binding antibodies. The common frequency for the variable portion of the heavy chain (VH) of the PC-specific antibodies is found to be at f = 0.37 Hz. The VH sequences of the PC-binding antibodies exhibit three subsites for the PC moiety where hypervariable region 2 (CDR2) plays a role in the interaction with the phosphate group. Mutations in this VH region have an impact on the ability of mutant variants to bind PC and its carrier molecule, as well as on the characteristic frequency shift toward f = 0.12 Hz for mutants failing to bind both hapten and carrier. The VH sequence of mutants that retain the ability to bind PC still shows f = 0.37 Hz, suggesting that this frequency determines PC binding. However, this statement was not confirmed as mutation in another PC subsite impairs PC binding but retains both the phosphate-group recognition and the frequency at f = 0.37 Hz. Herein, this finding is discussed to promote the idea that the VH sequence of the PC-binding antibodies encodes the subsite for phosphate-group binding as a dominant functional activity and that only CDR2 of the T15-idiotype antibodies together with FR3 region form an autonomous self-association function represented by the T15VH50-73 peptide with f = 0.37±0.05 Hz. Thus, these data confirmed that T15VH50-73 peptide might be used in superantibody technology.

Naturally occurring V region connected antibodies inhibit anti-dsDNA antibody reactivity with dsDNA.

The production of autoantibodies against a vast array of self antigens, most notably double stranded (ds) DNA, characterized systemic lupus erythematosus (SLE). The purpose of this work is to study specific Ig fractions isolated from normal human serum (NHS) and their effect on the binding of anti-double-stranded deoxyribonucleic acid (dsDNA) antibodies (Abs) to dsDNA. A fraction named immunoglobulin G (IgG)-reactive IgG was purified from total NHS IgG by absorption onto (CNBr)-activated Sepharose 4B linked to intact IgG molecules (IgG-Sepharose column). IgG-reactive IgG was co-incubated with systemic lupus erythematosus (SLE) patient's serum and binding of the anti-dsDNA Abs to dsDNA was measured by enzyme-linked immunosorbent assay (ELISA). Co-incubation of SLE patient's serum with IgG-reactive IgG resulted in a dose-dependent reduction in binding of anti-dsDNA Abs to dsDNA. A reduction greater than 70% was observed at a concentration of 300 µg of IgG-reactive IgG per mL of a 400-fold diluted SLE patient's serum whereas total NHS IgG, at the same concentration, resulted in a 10% reduction in binding. The purification process used to isolate IgG-reactive IgG was based on interactions between intact Ig rather than on interactions between F(ab')(2) portions. IgG(2) is the predominant immunoglobulin (Ig) subclass in IgG-reactive IgG. Thus, IgG(2) might have an important role in the connectivity characteristics of NHS IgG. The capacity of IgG-reactive IgG to inhibit anti-DNA Ab binding to dsDNA may have potential application in the treatment of SLE. This targeted biological approach may provide an alternative strategy to immunosuppressants.

Immunoglobulin G-reactive antibodies from sera of healthy individuals enriched in IgG2--therapeutic potential in HIV-1 infection.

OBJECTIVE: To characterize the functional properties of natural autoantibodies capable of preventing in vitro infection by HIV-1, present in normal human serum (NHS), and denoted as IgG-reactive antibodies. METHODS: IgG-reactive antibodies were affinity purified both from normal human serum (NHS) and from a GammaBind G Sepharose Flowthrough (GBF) fraction of NHS by affinity chromatography on IgG coupled to CNBr-activated Sepharose (IgG-Sepharose). RESULTS: The GBF fraction was shown, by Capture ELISA relative to isotype-matched standards, to contain in addition to IgM and IgA isotypes, a low but constant level of IgG isotype. About 15% of the GBF fraction's IgG, compared to only about 0.3% of the NHS IgG, was affinity purified on IgG-Sepharose. On IgG subclass analysis, in contrast to the characteristic dominance of IgG1 in pooled NHS, the IgG-reactive antibodies obtained from NHS and from the GBF fraction each showed a dominance of IgG2. Western blot analysis confirmed the abundance of IgG2, a major IgG subclass reactive against carbohydrate antigens, and showed the presence of IgG2 dimers. The IgG-reactive antibodies separated from the GBF fraction were able to neutralize HIV-1(BaL) strain with approaching 100% and 80% effectiveness at 2 microg/ml and 0.6 microg/ml, respectively, as well as the primary isolates HIV-1(NDK) (X4-tropic isolate) and HIV-1(JR-CSF) (R5-tropic isolate) with an IC50 between 0.4 microg/ml and 1.8 microg/ml for two different preparations. CONCLUSION: These findings further support our previous proposal for IgG-reactive antibody preparations to be used in the treatment of HIV-1 infected individuals.

Anti-IgG antibodies from sera of healthy individuals neutralize HIV-1 primary isolates.

Immunoglobulins (Ig) of pooled healthy human sera were purified by affinity chromatography based on their reactivity with human IgG. This Ig fraction represent connected, natural antibodies (NAbs) and here are denoted as anti-IgG antibodies. The data revealed that IgG, IgA and IgM isotypes are constituents of anti-IgG fraction. The ability of anti-IgG antibodies to prevent infection of PBMC by HIV-1 was demonstrated. They exhibited different neutralizing activity depending on the phenotype of the tested virus. The efficacy of neutralization was comparable to monoclonal antibodies (MAbs) IgG1b12 at least for the HIV-1 (92HT593B) strain. These studies suggest that connected antibodies thus, constituents of immune network, could prevent infection by HIV-1. NAbs as essential components of therapeutic molecules of intravenous Ig (IVIg) have a beneficial effect on variety of immunological disorders by affecting the structure, function and dynamics of the immune network. Since, hallmark of HIV-1 infection are immunological disorders we hypothesizes that they might be corrected to some extend by anti-IgG antibodies.

Antibodies reactive with C-terminus of the second conserved region of HIV-1gp120 as possible prognostic marker and therapeutic agent for HIV disease.

It has been reported that antibodies reactive with peptide RSANFTDNAKTIIVQLNQSVEIN (peptide NTM) derived from the C-terminus of the second conserved domain of HIV-1 envelope glycoprotein gp120 could represent an important factor in control of the HIV disease. In order to check this notion we (i) tested reactivity with peptide NTM serum samples collected from 310 consecutive HIV-1 infected patients with a CD4(+) lymphocyte count ranging from 10 to 800/microL and (ii) performed the longitudinal study that included 107 sera samples collected from 29 HIV patients. Results of these studies demonstrated correlation between presence of anti-NTM antibodies in sera of HIV patients and disease progression measured by the CD4(+) cell count. Based on these findings we proposed the anti-NTM antibodies as useful prognostic marker for HIV disease.

Molecular makeup of HIV-1 envelope protein.

A broad range of structural, functional, and immunological similarities between HIV-1 gp120 and human proteins, especially those participating in immune responses, highlight gp120 as a pleiotropic protein that can in different ways affect many important functions of the human immune system. Here we described some of these properties of HIV-1 gp120 that represent the main obstacle in the development of effective and safe AIDS vaccine.

Application of VIP/NTM-reactive natural antibodies in therapy of HIV disease.

In sera of HIV-infected individuals natural antibodies recognizing nonimmunogenic C-terminal domain of the second conserved region of HIV-1 gp120 and the vasoactive intestinal peptide (VIP) were identified. It has been demonstrated that these antibodies are significantly more prevalent in asymptomatic carriers than in AIDS patients and that their titer strongly correlates with disease progression. These findings point out the VIP/C2-reactive natural antibodies as an important agent for immunotherapy of HIV disease.

HIV-1 gp120 and immune network.

It has been demonstrated that the immunodominant V3 loop of HIV-1 gp120 and its flanking regions bear sequence and structural homology to the framework and complementarity-determining regions of human immunoglobulins. It has been proposed that the Ig-like domain of gp120 might encode idiotypes and in this way permit HIV-1 entry into the immune regulatory network. This notion is strongly supported by results demonstrating that the anti-V3 loop and anti-Ig antibodies of healthy individuals share complementary structure and that V3 reactive antibodies are present in HIV-negative sera. This might be the mechanism by which HIV induces immunological abnormalities, and it should be taken into consideration in AIDS vaccine development.