Circulating anti-citrullinated protein antibodies containing secretory component are prognostic for arthritis onset in at-risk patients.

Autoantibodies related to rheumatoid arthritis (RA), such as anti-citrullinated protein antibodies (ACPA), are often detectable in the preclinical period years before arthritis onset. However, events triggering arthritis development remain incompletely known. We aimed to determine whether ACPA isotype levels are prognostic for arthritis development in patients presenting with immunoglobulin (Ig)G ACPA and musculoskeletal pain. Study participants (n = 82) had musculoskeletal pain of any sort and duration and a positive IgG ACPA test. None of the patients had arthritis upon clinical examination at baseline, but during follow-up (mean = 6 years), 48% developed at least one arthritic joint. IgG, IgA, IgM and secretory component (SC)-containing ACPA was measured in longitudinally collected serum samples. Cox regression analysis was performed to test the prognostic value of baseline antibody levels and changes over time. All analysed ACPA isotype levels were associated with arthritis development in univariable Cox regression analysis. In multivariable analysis, baseline SC ACPA levels were independently prognostic for arthritis development in multivariable analysis [hazard ratio (HR) = 1·006, 95% confidence interval (CI) = 1·001-1·010, P = 0·012]. There were no significant changes in ACPA isotype levels over time, and no significant association between changes over time and arthritis development. In this prospective longitudinal study, baseline serum SC ACPA levels, but neither IgG, IgA nor IgM ACPA are prognostic for future arthritis development. Repeated measurement of ACPA isotypes do not bring additional prognostic value. The results reinforce a mucosal connection in RA development and encourage further exploration of the mechanisms underlying secretory ACPA formation as a trigger for arthritis development.

Secretory antibodies to citrullinated peptides in plasma and saliva from rheumatoid arthritis patients and their unaffected first-degree relatives.

The aim of this study was to evaluate secretory antibodies to citrullinated proteins (ACPA) in plasma and immunoglobulin (Ig)A ACPA in saliva from patients with rheumatoid arthritis (RA) and their unaffected first-degree relatives (FDRs). Patients with RA (n = 194) and first-degree relatives unaffected by RA (n = 191) were recruited for analysis of secretory antibodies to second-generation cyclic citrullinated peptides (anti-CCP) in plasma. From a subpopulation (25 RA patients, 21 first-degree relatives and 11 controls), saliva samples were obtained for IgA anti-CCP analysis. The presence of secretory ACPA was compared between subject categories, and related to genetic and environmental risk factors. Secretory ACPA occurred in 37 (19%) plasma samples from patients with RA, but only in two (1%) of FDRs. IgA ACPA in saliva was found in three of 25 (12%) patients with RA, but not in any of the 21 FDRs (< 5%). No significant associations were seen between the presence of secretory ACPA and SE or smoking, either among RA patients or among FDRs. Despite occurring in 19% of RA plasma, secretory ACPA was rare in both saliva and plasma among FDRs, even among those positive for conventional ACPA of non-mucosal origin. Longitudinal studies are warranted to determine whether circulating secretory ACPA occurs before or in parallel with the development of clinical arthritis.

Changes in anti-citrullinated protein antibody isotype levels in relation to disease activity and response to treatment in early rheumatoid arthritis.

Rheumatoid arthritis (RA) is a chronic inflammatory disease where serum analysis of anti-citrullinated peptide/protein antibodies (ACPA) is an important diagnostic/prognostic tool. Levels and changes of ACPA in RA patients have been studied previously in relation to disease course and therapy response, but less is known regarding ACPA isotype changes in early RA. Hence, recent-onset RA patients (n = 231) were subjected to a 3-year clinical and radiological follow-up. Serum samples were serially collected and ACPA isotypes were analysed using the second-generation cyclic citrullinated peptide (CCP) as capture antigen. Changes in ACPA isotype levels and status were related to disease course and pharmacotherapy. At inclusion, 74% of the patients tested positive for ACPA IgG; 55% for immunoglobulin (Ig)A, 37% for secretory IgA (SIgA) and 35% for IgM. The proportion of positive patients decreased significantly at follow-up regarding ACPA SIgA, IgM and IgA. During the initial 3 months, reduction of the 28-joint disease activity score (DAS28) correlated with reduced levels of ACPA IgG (Rho = 0·242, P = 0·003), IgA (Rho = 0·260, P = 0·008), IgM (Rho = 0·457, P < 0·001) and SIgA (Rho = 0·402, P < 0·001). Levels of ACPA SIgA (P = 0·008) and IgM (P = 0·021) decreased significantly among patients with good response to treatment, which was not seen regarding ACPA IgA or IgG. Changes in ACPA isotype levels were not associated with radiographic damage. In conclusion, ACPA SIgA and IgM declined rapidly upon anti-rheumatic therapy and correlated with decreased disease activity in recent-onset RA. This may indicate that down-regulation of mucosal immunity to citrullinated proteins/peptides and recruitment of new B cells are key features of therapy responses in early RA.

Immunization with HIV-1 envelope T20-encoding DNA vaccines elicits cross-clade neutralizing antibody responses.

BACKGROUND: Genetic immunization is expected to induce the expression of antigens in a native form. The encoded peptide epitopes are presented on endogenous MHC molecules, mimicking antigen presentation during a viral infection. We have explored the potential of enfuvirtide (T20), a short HIV peptide with antiviral properties, to enhance immune response to HIV antigens. To generate an expression vector, the T20 sequence was cloned into a conventional plasmid, the novel minicircle construct, and a replicon plasmid. In addition, 3 conventional plasmids that express the envelope of HIV-1 subtypes A, B and C and contain T20 in their gp41 sequences were also tested. RESULTS: All combinations induced HIV-specific antibodies and cellular responses. The addition of T20 as a peptide and as an expression cassette in the 3 DNA vectors enhanced antibody responses. The highest anti-HIV-1 Env titers were obtained by the replicon T20 construct. This demonstrates that besides its known antiviral activity, T20 promotes immune responses. We also confirm that the combination of slightly divergent antigens improves immune responses. CONCLUSIONS: The antiretroviral T20 HIV-1 sequence can be used as an immunogen to elicit binding and neutralizing antibodies against HIV-1. These, or similarly modified gp41 genes/peptides, can be used as priming or boosting components for induction of broadly neutralizing anti-HIV antibodies. Future comparative studies will reveal the optimal mode of T20 administration.

HIVIS-DNA or HIVISopt-DNA priming followed by CMDR vaccinia-based boosts induce both humoral and cellular murine immune responses to HIV.

BACKGROUND: In order to develop a more effective prophylactic HIV-1 vaccine it is important optimize the components, improve Envelope glycoprotein immunogenicity as well as to explore prime-boost immunization schedules. It is also valuable to include several HIV-1 subtype antigens representing the world-wide epidemic. METHODS: HIVIS-DNA plasmids which include Env genes of subtypes A, B and C together with Gag subtypes A and B and RTmut/Rev of subtype B were modified as follows: the Envelope sequences were shortened, codon optimized, provided with an FT4 sequence and an immunodominant region mutated. The reverse transcriptase (RT) gene was shortened to contain the most immunogenic N-terminal fragment and fused with an inactivated viral protease vPR gene. HIVISopt-DNA thus contains fewer plasmids but additional PR epitopes compared to the native HIVIS-DNA. DNA components were delivered intradermally to young Balb/c mice once, using a needle-free Biojector® immediately followed by dermal electroporation. Vaccinia-based MVA-CMDR boosts including Env gene E and Gag-RT genes A were delivered intramuscularly by needle, once or twice. RESULTS: Both HIVIS-DNA and HIVISopt-DNA primed humoral and cell mediated responses well. When boosted with heterologous MVA-CMDR (subtypes A and E) virus inhibitory neutralizing antibodies were obtained to HIV-1 subtypes A, B, C and AE. Both plasmid compositions boosted with MVA-CMDR generated HIV-1 specific cellular responses directed against HIV-1 Env, Gag and Pol, as measured by IFNγ ELISpot. It was shown that DNA priming augmented the vector MVA immunological boosting effects, the HIVISopt-DNA with a trend to improved (Env) neutralization, the HIVIS-DNA with a trend to better (Gag) cell mediated immune reponses. CONCLUSIONS: HIVIS-DNA was modified to obtain HIVISopt-DNA that had fewer plasmids, and additional epitopes. Even with one DNA prime followed by two MVA-CMDR boosts, humoral and cell-mediated immune responses were readily induced by priming with either DNA construct composition. Priming by HIV-DNA augmented neutralizing antibody responses revealed by boosting with the vaccinia-based heterologous sequences. Cellular and antibody responses covered selected strains representing HIV-1 subtypes A, B, C and CRF01_AE. We assume this is related to the inclusion of heterologous full genes in the vaccine schedule.

The rationale behind a vaccine based on multiple HIV antigens.

The viral diversity of HIV-1 is likely to require a vaccine strategy that induces broad cellular and humoral anti-HIV-1 immunity. Our strategy is based on multiple HIV-1 DNA immunogens together with adjuvant recombinant granulocyte-macrophage stimulating factor. This article describes pre-clinical and clinical work preceding the initiation of clinical HIV-1 phase I/II trials.

Simultaneous search for multiple QTL using the global optimization algorithm DIRECT.

MOTIVATION: A simultaneous search is necessary for maximizing the power to detect epistatic quantitative trait loci (QTL). The computational complexity demands that the traditional exhaustive search be replaced by a more efficient global optimization algorithm. RESULTS: We have the previously known algorithm adapted DIRECT, to the problem of simultaneous mapping of multiple QTL. We have compared DIRECT with standard exhaustive search and a genetic algorithm previously used for QTL mapping in two dimensions. In all two- and three-QTL test cases, DIRECT accurately finds the global optimum two to four orders of magnitude faster than when using an exhaustive search, and one order of magnitude faster than when using the genetic algorithm. Thus, randomization testing for determining empirical significance thresholds for at least three QTL is made feasible by the use of DIRECT. AVAILABILITY: The code of the prototype implementation is available at http://user.it.uu.se/~kl/qtl_software.html

Genetic immunization is augmented by murine polyomavirus VP1 pseudocapsids.

To improve immune responses induced by DNA immunization, murine polyomavirus major capsid protein (VP1) pseudocapsids were complexed with a DNA plasmid encoding the p37 (p24 and p17) nucleocapsid proteins of the human immunodeficiency virus type 1 (HIV-1). A 10-fold increase in antibody titer was noted in mice given DNA plasmid together with VP1 pseudocapsids in comparison to animals that received DNA plasmid alone. Cell mediated responses to HIV-1 p24 occurred, but were not significantly augmented by delivering the DNA as a VP1 complex. We have consequently for the first time shown a carrier/adjuvant effect of polyomavirus pseudocapsids that strongly increased the humoral immune response in DNA immunization.

HIV subtypes and recombination strains--strategies for induction of immune responses in man.

Clinical and experimental studies of HIV-1 subcomponents were made in order to increase their immunogenicity. HIV subtype envelopes A, B and C have been compared and a detailed analysis made by peptides of the coreceptor-ligand interactions. We identified a direct interaction between HIV-1 envelope and a cellular receptor at the amino acid level. Both the viral subtype and its tropism appeared to influence inhibition of infection. Genetic immunization induced new cytotoxic responses while proteins appeared to efficiently boost previous responses. One HIV-1 subtype B antigen was strongly immunogenic in a human immunotherapeutic trial and permitted better survival at 2 years of the study in patients with poor prognosis.

Sensitivity of an empirical affinity scoring function to changes in receptor-ligand complex conformations.

A combination of empirical scoring and conformational sampling for ligand binding affinity prediction is examined. The behaviour of a scoring function with respect to the sensitivity to conformational changes is investigated using ensembles of structures generated by molecular dynamics simulation. The correlation between the calculated score and the coordinate deviation from the experimental structure is clear for the complex of arabinose with arabinose-binding protein, which is dominated by hydrogen bond interactions, while the score calculated for the hydrophobic complex between retinol and retinol binding protein is rather insensitive to ligand conformational changes. For typical ensembles of structures generated by molecular dynamics at 300 K, the variation of the calculated score is considerably smaller than that of the underlying molecular mechanics interaction energies.

Computational modelling of inhibitor binding to human thrombin.

Thrombin is an essential protein involved in blood clot formation and an important clinical target, since disturbances of the coagulation process cause serious cardiovascular diseases such as thrombosis. Here we evaluate the performance of a molecular dynamics based method for predicting the binding affinities of different types of human thrombin inhibitors. For a series of eight ligands the method ranks their relative affinities reasonably well. The binding free energy difference between high and low affinity representatives in the test set is quantitatively reproduced, as well as the stereospecificity for a chiral inhibitor. The original parametrisation of this linear interaction energy method requires the addition of a constant energy term in the case of thrombin. This yields a mean unsigned error of 0.68 kcal/mol for the absolute binding free energies. This type of approach is also useful for elucidating three-dimensional structure-activity relationships in terms of microscopic interactions of the ligands with the solvated enzyme.

Effective construction of DNA vaccines against variable influenza genes by homologous recombination.

We demonstrate the potential of cloning by homologous recombination as a rapid method to construct DNA molecules encoding newly developing hemagglutinins (HA) of influenza A virus. The variable parts of the HA genes were cloned into a basic construct containing the HA gene from an H3N2 strain. The recombinant DNAs thus created encode different variable domains with neutralising epitopes from four recently circulating influenza A H3 strains. The technology allows rapid production of DNA constructs for vaccines that can induce antibody and, particularly, cellular immune responses. These new constructs were also capable of conferring protection to challenge in mice. The technology may hence be a valuable tool for rapid adaptation of influenza vaccines to changes in the circulating influenza strains.