David Davis

Analysis of memory B cell responses and isolation of novel monoclonal antibodies with neutralizing breadth from HIV-1-infected individuals.

Background The isolation of human monoclonal antibodies (mAbs) that neutralize a broad spectrum of primary HIV-1 isolates and the characterization of the human neutralizing antibody B cell response to HIV-1 infection are important goals that are central to the design of an effective antibody-based vaccine. Methods and Findings We immortalized IgG+ memory B cells from individuals infected with diverse clades of HIV-1 and selected on the basis of plasma neutralization profiles that were cross-clade and relatively potent. Culture supernatants were screened using various recombinant forms of the envelope glycoproteins (Env) in multiple parallel assays. We isolated 58 mAbs that were mapped to different Env surfaces, most of which showed neutralizing activity. One mAb in particular (HJ16) specific for a novel epitope proximal to the CD4 binding site on gp120 selectively neutralized a multi-clade panel of Tier-2 HIV-1 pseudoviruses, and demonstrated reactivity that was comparable in breadth, but distinct in neutralization specificity, to that of the other CD4 binding site-specific neutralizing mAb b12. A second mAb (HGN194) bound a conserved epitope in the V3 crown and neutralized all Tier-1 and a proportion of Tier-2 pseudoviruses tested, irrespective of clade. A third mAb (HK20) with broad neutralizing activity, particularly as a Fab fragment, recognized a highly conserved epitope in the HR-1 region of gp41, but showed striking assay-dependent selectivity in its activity. Conclusions This study reveals that by using appropriate screening methods, a large proportion of memory B cells can be isolated that produce mAbs with HIV-1 neutralizing activity. Three of these mAbs show unusual breadth of neutralization and therefore add to the current panel of HIV-1 neutralizing antibodies with potential for passive protection and template-based vaccine design.

Potent and broad neutralization of HIV-1 by a llama antibody elicited by immunization

A heavy chain–only antibody isolated from a llama repeatedly immunized with trimeric HIV-1 Env neutralizes 96% of tested HIV-1 strains.

A gp41 MPER-specific llama VHH requires a hydrophobic CDR3 for neutralization but not for antigen recognition.

The membrane proximal external region (MPER) of the HIV-1 glycoprotein gp41 is targeted by the broadly neutralizing antibodies 2F5 and 4E10. To date, no immunization regimen in animals or humans has produced HIV-1 neutralizing MPER-specific antibodies. We immunized llamas with gp41-MPER proteoliposomes and selected a MPER-specific single chain antibody (VHH), 2H10, whose epitope overlaps with that of mAb 2F5. Bi-2H10, a bivalent form of 2H10, which displayed an approximately 20-fold increased affinity compared to the monovalent 2H10, neutralized various sensitive and resistant HIV-1 strains, as well as SHIV strains in TZM-bl cells. X-ray and NMR analyses combined with mutagenesis and modeling revealed that 2H10 recognizes its gp41 epitope in a helical conformation. Notably, tryptophan 100 at the tip of the long CDR3 is not required for gp41 interaction but essential for neutralization. Thus bi-2H10 is an anti-MPER antibody generated by immunization that requires hydrophobic CDR3 determinants in addition to epitope recognition for neutralization similar to the mode of neutralization employed by mAbs 2F5 and 4E10.

HIV-1 vaccine-induced immune responses which correlate with protection from SHIV infection: compiled preclinical efficacy data from trials with ten different HIV-1 vaccine candidates.

The specific immune mechanisms necessary and/or sufficient to elicit HIV-vaccine protection remain undefined. Utilising the SHIV rhesus macaque model the immunogenicity as well as the efficacy of ten different HIV-1 vaccine candidates was evaluated. Comparison of the immune responses induced, with the ability of the vaccine to protect from SHIV infection provided a means to determine which type of immune responses were necessary for protection. Vaccine candidates included VLPs, DNA, subunit protein with novel adjuvant formulations, ISCOMs and pox-virus vectors. Protection from SHIV infection was achieved in approximately half of the animals which received a primary intravenous cell-free challenge. The presence of CTL in the absence of other effector responses did not correlate with protection from this route and type of challenge. Virus neutralising antibodies (Nab) appeared to be necessary but alone were insufficient for protection. If Ag-specific IFN-gamma and/or IL-4 as well as lymphoproliferative (LP) responses were found with the lack of a detectable IL-2 response, then protection was not observed. Immunity correlated with the magnitude of Nab responses, beta-chemokines and as well as balanced, qualitative T-helper responses.

Systemic neutralizing antibodies induced by long interval mucosally primed systemically boosted immunization correlate with protection from mucosal SHIV challenge

Immune correlates of vaccine protection from HIV-1 infection would provide important milestones to guide HIV-1 vaccine development. In a proof of concept study using mucosal priming and systemic boosting, the titer of neutralizing antibodies in sera was found to correlate with protection of mucosally exposed rhesus macaques from SHIV infection. Mucosal priming consisted of two sequential immunizations at 12-week intervals with replicating host range mutants of adenovirus type 5 (Ad5hr) expressing the HIV-1(89.6p) env gene. Following boosting with either heterologous recombinant protein or alphavirus replicons at 12-week intervals animals were intrarectally exposed to infectious doses of the CCR5 tropic SHIV(SF162p4). Heterologous mucosal prime systemic boost immunization elicited neutralizing antibodies (Nabs), antibody-dependent cytotoxicity (ADCC), and specific patterns of antibody binding to envelope peptides. Vaccine induced protection did not correlate with the type of boost nor T-cell responses, but rather with the Nab titer prior to exposure.

Rheumatic heart disease: III. Embolic manifestations☆☆☆

Abstract 1. 1. Infarction of one or more organs occurred in 73 cases, an incidence of 45 per cent, of a group of 164 cases in which death was caused by rheumatic heart disease. In 41 instances one organ, in 22 instances two organs, and in 10 instances three or more organs were involved by single or multiple infarcts. The organs involved in order of frequency were: lungs 31, brain 28, kidneys 25, spleen 18, extremities 10, intestines 5, and liver 1. 2. 2. A conservative estimate revealed that in 34 cases, or 21 per cent of the total group, embolism played a chief or contributing part in the death of the patients. Embolism plays, therefore, a significant role in the causation of death in rheumatic heart disease. 3. 3. A statistical consideration suggests that auricular fibrillation rather than active rheumatic endocarditis of the auricles plays the primary etiological role in the formation of auricular thrombi. 4. 4. Rheumatic heart disease, more than any other type of heart disease, is responsible for embolic manifestations. 5. 5. Clinical considerations bearing on embolic manifestations are discussed.

Potent Immune Responses in Rhesus Macaques Induced by Nonviral Delivery of a Self-amplifying RNA Vaccine Expressing HIV Type 1 Envelope With a Cationic Nanoemulsion

Self-amplifying messenger RNA (mRNA) of positive-strand RNA viruses are effective vectors for in situ expression of vaccine antigens and have potential as a new vaccine technology platform well suited for global health applications. The SAM vaccine platform is based on a synthetic, self-amplifying mRNA delivered by a nonviral delivery system. The safety and immunogenicity of an HIV SAM vaccine encoding a clade C envelope glycoprotein formulated with a cationic nanoemulsion (CNE) delivery system was evaluated in rhesus macaques. The HIV SAM vaccine induced potent cellular immune responses that were greater in magnitude than those induced by self-amplifying mRNA packaged in a viral replicon particle (VRP) or by a recombinant HIV envelope protein formulated with MF59 adjuvant, anti-envelope binding (including anti-V1V2), and neutralizing antibody responses that exceeded those induced by the VRP vaccine. These studies provide the first evidence in nonhuman primates that HIV vaccination with a relatively low dose (50 µg) of formulated self-amplifying mRNA is safe and immunogenic.

Differences in early virus loads with different phenotypic variants of HIV-1 and SIV(cpz) in chimpanzees.

ObjectiveA comparative study of the replication kinetics of different HIV-1 variants (including SIVcpz) was undertaken to determine which viral characteristics were associated with sustained plasma viraemia in chimpanzees. DesignPlasma samples from chimpanzees infected with six different HIV-1 clade B isolates were compared with plasma samples from SIVcpz−ant-infected chimpanzees. MethodsA pan-clade quantitative competitive reverse transcriptase–polymerase chain reaction assay was developed based on conserved primer sequences recognizing M, N and O human lentiviruses as well as different SIVcpz isolates. ResultsImportant differences between early kinetics in the human lentivirus isolates as well as compared with the chimpanzee isolate SIVcpz−ant were observed. R5-dependent non-syncytium-inducing (NSI) isolates (5016, Ba-L, SIVcpz) were found to have relatively higher viral loads than the syncytium-inducing (SI), X4-dependent primary (SF2), T cell-adapted (IIIB) or X4/R5 (Han2, DH12) SI primary isolates. ConclusionInfection of chimpanzees with NSI R5-utilizing isolates correlated with persistent viraemia (approximately 104 RNA equivalents/ml) in contrast to transient viraemia observed after infection with SI X4-utilizing isolates.

The N276 glycosylation site is required for HIV-1 neutralization by the CD4 binding site specific HJ16 monoclonal antibody

Immunogen design for HIV-1 vaccines could be based on epitope identification of naturally occurring neutralizing antibodies in infected patients. A tier 2 neutralizing monoclonal antibody (mAb), HJ16 recognizes a new epitope in the CD4 binding site (CD4bs) region that only partially overlaps with the b12 epitope. We aimed to identify the critical binding site by resistance induction in a sensitive primary CRF02_AG strain. In four independent dose-escalation studies, the N276D mutation was consistently the only alteration found and it was confirmed to be responsible for resistance to HJ16 by site-directed mutagenesis in envelopes (envs) of the homologous CRF02_AG, as well as of a subtype A and a subtype C primary isolate. This mutation removes an N-linked glycosylation site. The effect of N276D was very selective, as it failed to confer resistance to a range of other entry inhibitors. Remarkably, sensitivity to the CD4bs VRC01 and VRC03 mAbs was increased in the N276D mutated viruses. These data indicate that binding of the CD4bs specific HJ16 mAb critically depends on the interaction with the N276-glycan, thus indicating that HJ16 is the first glycan dependent CD4bs-specific mAb.

A recombinant prime, peptide boost vaccination strategy can focus the immune response on to more than one epitope even though these may not be immunodominant in the complex immunogen.

Rhesus monkeys were successfully vaccinated using a strategy of priming with a candidate envelope subunit vaccine and boosting with synthetic peptides. Priming was carried out with recombinant HIV-1 SF2 envelope glycoprotein incorporated into ISCOMs, following the attachment of a lipid tail. Peptides, covalently linked to ISCOMs, representing linear sequences with the V2 and V3 regions, were used to boost functional antibodies-to neutralizing epitopes in both of these regions. Injections with these peptide formulations substantially increased the titre of serum neutralizing antibodies from low or undetectable levels. In addition to completely neutralizing the homologous HIV-1 SF2 strain, these sera also neutralized the escape variant, HIV-1 SF13. However, no antibodies were boosted which could compete with human, neutralizing monoclonal antibodies recognising conformational epitopes. The peptides also boosted antibodies to a peptide whose sequence lies close to the V2 region neutralizing epitope but does not overlap with it. Importantly, the level of antibodies to an unrelated epitope associated with enhancement of HIV-1 SF13 continued to fall after the peptide boost. Successful protection against challenge with chimeric simian immunodeficiency virus expressing HIV-1 SF13 envelope glycoproteins (SHIV SF13) may be due to an increase in the ratio of neutralizing to enhancing antibodies by selectively boosting with peptides to critical neutralizing epitopes.