Optimal Combinations of Broadly Neutralizing Antibodies for Prevention and Treatment of HIV-1 Clade C Infection.

The identification of a new generation of potent broadly neutralizing HIV-1 antibodies (bnAbs) has generated substantial interest in their potential use for the prevention and/or treatment of HIV-1 infection. While combinations of bnAbs targeting distinct epitopes on the viral envelope (Env) will likely be required to overcome the extraordinary diversity of HIV-1, a key outstanding question is which bnAbs, and how many, will be needed to achieve optimal clinical benefit. We assessed the neutralizing activity of 15 bnAbs targeting four distinct epitopes of Env, including the CD4-binding site (CD4bs), the V1/V2-glycan region, the V3-glycan region, and the gp41 membrane proximal external region (MPER), against a panel of 200 acute/early clade C HIV-1 Env pseudoviruses. A mathematical model was developed that predicted neutralization by a subset of experimentally evaluated bnAb combinations with high accuracy. Using this model, we performed a comprehensive and systematic comparison of the predicted neutralizing activity of over 1,600 possible double, triple, and quadruple bnAb combinations. The most promising bnAb combinations were identified based not only on breadth and potency of neutralization, but also other relevant measures, such as the extent of complete neutralization and instantaneous inhibitory potential (IIP). By this set of criteria, triple and quadruple combinations of bnAbs were identified that were significantly more effective than the best double combinations, and further improved the probability of having multiple bnAbs simultaneously active against a given virus, a requirement that may be critical for countering escape in vivo. These results provide a rationale for advancing bnAb combinations with the best in vitro predictors of success into clinical trials for both the prevention and treatment of HIV-1 infection.

Longitudinal Antigenic Sequences and Sites from Intra-Host Evolution (LASSIE) Identifies Immune-Selected HIV Variants.

Within-host genetic sequencing from samples collected over time provides a dynamic view of how viruses evade host immunity. Immune-driven mutations might stimulate neutralization breadth by selecting antibodies adapted to cycles of immune escape that generate within-subject epitope diversity. Comprehensive identification of immune-escape mutations is experimentally and computationally challenging. With current technology, many more viral sequences can readily be obtained than can be tested for binding and neutralization, making down-selection necessary. Typically, this is done manually, by picking variants that represent different time-points and branches on a phylogenetic tree. Such strategies are likely to miss many relevant mutations and combinations of mutations, and to be redundant for other mutations. Longitudinal Antigenic Sequences and Sites from Intrahost Evolution (LASSIE) uses transmitted founder loss to identify virus "hot-spots" under putative immune selection and chooses sequences that represent recurrent mutations in selected sites. LASSIE favors earliest sequences in which mutations arise. With well-characterized longitudinal Env sequences, we confirmed selected sites were concentrated in antibody contacts and selected sequences represented diverse antigenic phenotypes. Practical applications include rapidly identifying immune targets under selective pressure within a subject, selecting minimal sets of reagents for immunological assays that characterize evolving antibody responses, and for immunogens in polyvalent "cocktail" vaccines.

Impact of clade, geography, and age of the epidemic on HIV-1 neutralization by antibodies.

UNLABELLED: Neutralizing antibodies (nAbs) are a high priority for vaccines that aim to prevent the acquisition of HIV-1 infection. Vaccine effectiveness will depend on the extent to which induced antibodies neutralize the global diversity of circulating HIV-1 variants. Using large panels of genetically and geographically diverse HIV-1 Env-pseudotyped viruses and chronic infection plasma samples, we unambiguously show that cross-clade nAb responses are commonly induced in response to infection by any virus clade. Nonetheless, neutralization was significantly greater when the plasma clade matched the clade of the virus being tested. This within-clade advantage was diminished in older, more-diverse epidemics in southern Africa, the United States, and Europe compared to more recent epidemics in Asia. It was most pronounced for circulating recombinant form (CRF) 07_BC, which is common in China and is the least-divergent lineage studied; this was followed by the slightly more diverse Asian CRF01_AE. We found no evidence that transmitted/founder viruses are generally more susceptible to neutralization and are therefore easier targets for vaccination than chronic viruses. Features of the gp120 V1V2 loop, in particular, length, net charge, and number of N-linked glycans, were associated with Env susceptibility and plasma neutralization potency in a manner consistent with neutralization escape being a force that drives viral diversification and plasma neutralization breadth. The overall susceptibility of Envs and potencies of plasma samples were highly predictive of the neutralization outcome of any single virus-plasma combination. These findings highlight important considerations for the design and testing of candidate HIV-1 vaccines that aim to elicit effective nAbs. IMPORTANCE: An effective HIV-1 vaccine will need to overcome the extraordinary variability of the virus, which is most pronounced in the envelope glycoproteins (Env), which are the sole targets for neutralizing antibodies (nAbs). Distinct genetic lineages, or clades, of HIV-1 occur in different locales that may require special consideration when designing and testing vaccines candidates. We show that nAb responses to HIV-1 infection are generally active across clades but are most potent within clades. Because effective vaccine-induced nAbs are likely to share these properties, optimal coverage of a particular clade or combination of clades may require clade-matched immunogens. Optimal within-clade coverage might be easier to achieve in regions such as China and Thailand, where the epidemic is more recent and the virus less diverse than in southern Africa, the United States, and Europe. Finally, features of the first and second hypervariable regions of gp120 (V1V2) may be critical for optimal vaccine design.

Antigenicity and immunogenicity of transmitted/founder, consensus, and chronic envelope glycoproteins of human immunodeficiency virus type 1.

Human immunodeficiency virus type 1 (HIV-1) vaccine development requires selection of appropriate envelope (Env) immunogens. Twenty HIV-1 Env glycoproteins were examined for their ability to bind human anti-HIV-1 monoclonal antibodies (MAbs) and then used as immunogens in guinea pigs to identify promising immunogens. These included five Envs derived from chronically infected individuals, each representing one of five common clades and eight consensus Envs based on these five clades, as well as the consensus of the entire HIV-1 M group, and seven transmitted/founder (T/F) Envs from clades B and C. Sera from immunized guinea pigs were tested for neutralizing activity using 36 HIV-1 Env-pseudotyped viruses. All Envs bound to CD4 binding site, membrane-proximal, and V1/V2 MAbs with similar apparent affinities, although the T/F Envs bound with higher affinity to the MAb 17b, a CCR5 coreceptor binding site antibody. However, the various Envs differed in their ability to induce neutralizing antibodies. Consensus Envs elicited the most potent responses, but neutralized only a subset of viruses, including mostly easy-to-neutralize tier 1 and some more-difficult-to-neutralize tier 2 viruses. T/F Envs elicited fewer potent neutralizing antibodies but exhibited greater breadth than chronic or consensus Envs. Finally, chronic Envs elicited the lowest level and most limited breadth of neutralizing antibodies overall. Thus, each group of Env immunogens elicited a different antibody response profile. The complementary benefits of consensus and T/F Env immunogens raise the possibility that vaccines utilizing a combination of consensus and T/F Envs may be able to induce neutralizing responses with greater breadth and potency than single Env immunogens.

Recurrent signature patterns in HIV-1 B clade envelope glycoproteins associated with either early or chronic infections.

Here we have identified HIV-1 B clade Envelope (Env) amino acid signatures from early in infection that may be favored at transmission, as well as patterns of recurrent mutation in chronic infection that may reflect common pathways of immune evasion. To accomplish this, we compared thousands of sequences derived by single genome amplification from several hundred individuals that were sampled either early in infection or were chronically infected. Samples were divided at the outset into hypothesis-forming and validation sets, and we used phylogenetically corrected statistical strategies to identify signatures, systematically scanning all of Env. Signatures included single amino acids, glycosylation motifs, and multi-site patterns based on functional or structural groupings of amino acids. We identified signatures near the CCR5 co-receptor-binding region, near the CD4 binding site, and in the signal peptide and cytoplasmic domain, which may influence Env expression and processing. Two signatures patterns associated with transmission were particularly interesting. The first was the most statistically robust signature, located in position 12 in the signal peptide. The second was the loss of an N-linked glycosylation site at positions 413-415; the presence of this site has been recently found to be associated with escape from potent and broad neutralizing antibodies, consistent with enabling a common pathway for immune escape during chronic infection. Its recurrent loss in early infection suggests it may impact fitness at the time of transmission or during early viral expansion. The signature patterns we identified implicate Env expression levels in selection at viral transmission or in early expansion, and suggest that immune evasion patterns that recur in many individuals during chronic infection when antibodies are present can be selected against when the infection is being established prior to the adaptive immune response.

Genetic signatures in the envelope glycoproteins of HIV-1 that associate with broadly neutralizing antibodies.

A steady increase in knowledge of the molecular and antigenic structure of the gp120 and gp41 HIV-1 envelope glycoproteins (Env) is yielding important new insights for vaccine design, but it has been difficult to translate this information to an immunogen that elicits broadly neutralizing antibodies. To help bridge this gap, we used phylogenetically corrected statistical methods to identify amino acid signature patterns in Envs derived from people who have made potently neutralizing antibodies, with the hypothesis that these Envs may share common features that would be useful for incorporation in a vaccine immunogen. Before attempting this, essentially as a control, we explored the utility of our computational methods for defining signatures of complex neutralization phenotypes by analyzing Env sequences from 251 clonal viruses that were differentially sensitive to neutralization by the well-characterized gp120-specific monoclonal antibody, b12. We identified ten b12-neutralization signatures, including seven either in the b12-binding surface of gp120 or in the V2 region of gp120 that have been previously shown to impact b12 sensitivity. A simple algorithm based on the b12 signature pattern was predictive of b12 sensitivity/resistance in an additional blinded panel of 57 viruses. Upon obtaining these reassuring outcomes, we went on to apply these same computational methods to define signature patterns in Env from HIV-1 infected individuals who had potent, broadly neutralizing responses. We analyzed a checkerboard-style neutralization dataset with sera from 69 HIV-1-infected individuals tested against a panel of 25 different Envs. Distinct clusters of sera with high and low neutralization potencies were identified. Six signature positions in Env sequences obtained from the 69 samples were found to be strongly associated with either the high or low potency responses. Five sites were in the CD4-induced coreceptor binding site of gp120, suggesting an important role for this region in the elicitation of broadly neutralizing antibody responses against HIV-1.

Breadth of human immunodeficiency virus-specific neutralizing activity in sera: clustering analysis and association with clinical variables.

Induction of antibodies that neutralize a broad range of human immunodeficiency virus type 1 (HIV-1) isolates is a major goal of vaccine development. To study natural examples of broad neutralization, we analyzed sera from 103 HIV-1-infected subjects. Among progressor patients, 20% of sera neutralized more than 75% of a panel of 20 diverse viral isolates. Little activity was observed in sera from long-term nonprogressors (elite controllers). Breadth of neutralization was correlated with viral load, but not with CD4 count, history of past antiretroviral use, age, gender, race/ethnicity, or route of exposure. Clustering analysis of sera by a novel method identified a statistically robust subgrouping of sera that demonstrated broad and potent neutralization activity.

Tiered categorization of a diverse panel of HIV-1 Env pseudoviruses for assessment of neutralizing antibodies.

The restricted neutralization breadth of vaccine-elicited antibodies is a major limitation of current human immunodeficiency virus-1 (HIV-1) candidate vaccines. In order to permit the efficient identification of vaccines with enhanced capacity for eliciting cross-reactive neutralizing antibodies (NAbs) and to assess the overall breadth and potency of vaccine-elicited NAb reactivity, we assembled a panel of 109 molecularly cloned HIV-1 Env pseudoviruses representing a broad range of genetic and geographic diversity. Viral isolates from all major circulating genetic subtypes were included, as were viruses derived shortly after transmission and during the early and chronic stages of infection. We assembled a panel of genetically diverse HIV-1-positive (HIV-1(+)) plasma pools to assess the neutralization sensitivities of the entire virus panel. When the viruses were rank ordered according to the average sensitivity to neutralization by the HIV-1(+) plasmas, a continuum of average sensitivity was observed. Clustering analysis of the patterns of sensitivity defined four subgroups of viruses: those having very high (tier 1A), above-average (tier 1B), moderate (tier 2), or low (tier 3) sensitivity to antibody-mediated neutralization. We also investigated potential associations between characteristics of the viral isolates (clade, stage of infection, and source of virus) and sensitivity to NAb. In particular, higher levels of NAb activity were observed when the virus and plasma pool were matched in clade. These data provide the first systematic assessment of the overall neutralization sensitivities of a genetically and geographically diverse panel of circulating HIV-1 strains. These reference viruses can facilitate the systematic characterization of NAb responses elicited by candidate vaccine immunogens.

Highly complex neutralization determinants on a monophyletic lineage of newly transmitted subtype C HIV-1 Env clones from India.

Little is known about the neutralization properties of HIV-1 in India to optimally design and test vaccines. For this reason, a functional Env clone was obtained from each of ten newly acquired, heterosexually transmitted HIV-1 infections in Pune, Maharashtra. These clones formed a phylogenetically distinct genetic lineage within subtype C. As Env-pseudotyped viruses the clones were mostly resistant to IgG1b12, 2G12 and 2F5 but all were sensitive to 4E10. When compared to a large multi-subtype panel of Env-pseudotyped viruses (subtypes B, C and CRF02_AG) in neutralization assays with a multi-subtype panel of HIV-1-positive plasma samples, the Indian Envs were remarkably complex. With the exception of the Indian Envs, results of a hierarchical clustering analysis showed a strong subtype association with the patterns of neutralization susceptibility. From these patterns we were able to identify 19 neutralization cluster-associated amino acid signatures in gp120 and 14 signatures in the ectodomain and cytoplasmic tail of gp41. We conclude that newly transmitted Indian Envs are antigenically complex in spite of close genetic similarity. Delineation of neutralization-associated amino acid signatures provides a deeper understanding of the antigenic structure of HIV-1 Env.

The global circulation of seasonal influenza A (H3N2) viruses.

Antigenic and genetic analysis of the hemagglutinin of approximately 13,000 human influenza A (H3N2) viruses from six continents during 2002-2007 revealed that there was continuous circulation in east and Southeast Asia (E-SE Asia) via a region-wide network of temporally overlapping epidemics and that epidemics in the temperate regions were seeded from this network each year. Seed strains generally first reached Oceania, North America, and Europe, and later South America. This evidence suggests that once A (H3N2) viruses leave E-SE Asia, they are unlikely to contribute to long-term viral evolution. If the trends observed during this period are an accurate representation of overall patterns of spread, then the antigenic characteristics of A (H3N2) viruses outside E-SE Asia may be forecast each year based on surveillance within E-SE Asia, with consequent improvements to vaccine strain selection.

Influenza vaccine strain selection and recent studies on the global migration of seasonal influenza viruses.

Annual influenza epidemics in humans affect 5-15% of the population, causing an estimated half million deaths worldwide per year [Stohr K. Influenza-WHO cares. Lancet Infectious Diseases 2002;2(9):517]. The virus can infect this proportion of people year after year because the virus has an extensive capacity to evolve and thus evade the immune response. For example, since the influenza A(H3N2) subtype entered the human population in 1968 the A(H3N2) component of the influenza vaccine has had to be updated almost 30 times to track the evolution of the viruses and remain effective. The World Health Organization Global Influenza Surveillance Network (WHO GISN) tracks and analyzes the evolution and epidemiology of influenza viruses for the primary purpose of vaccine strain selection and to improve the strain selection process through studies aimed at better understanding virus evolution and epidemiology. Here we give an overview of the strain selection process and outline recent investigations into the global migration of seasonal influenza viruses.

Mapping the antigenic and genetic evolution of influenza virus.

The antigenic evolution of influenza A (H3N2) virus was quantified and visualized from its introduction into humans in 1968 to 2003. Although there was remarkable correspondence between antigenic and genetic evolution, significant differences were observed: Antigenic evolution was more punctuated than genetic evolution, and genetic change sometimes had a disproportionately large antigenic effect. The method readily allows monitoring of antigenic differences among vaccine and circulating strains and thus estimation of the effects of vaccination. Further, this approach offers a route to predicting the relative success of emerging strains, which could be achieved by quantifying the combined effects of population level immune escape and viral fitness on strain evolution.

Recognition of homo- and heterosubtypic variants of influenza A viruses by human CD8+ T lymphocytes.

In the present study, the recognition of epitope variants of influenza A viruses by human CTL was investigated. To this end, human CD8(+) CTL clones, specific for natural variants of the HLA-B*3501-restricted epitope in the nucleoprotein (NP(418-426)), were generated. As determined in (51)Cr release assays and by flow cytometry with HLA-B*3501-peptide tetrameric complexes, CTL clones were found to be specific for epitopes within one subtype or cross-reactive with heterosubtypic variants of the epitope. Using eight natural variants of the epitope, positions in the 9-mer important for T cell recognition and involved in escape from CTL immunity were identified and visualized using multidimensional scaling. It was shown that positions 4 and 5 in the 9-mer epitope were important determinants of T cell specificity. The in vivo existence of CD8(+) cells cross-reactive with homo- and heterosubtypic variants of the epitope was further confirmed using polyclonal T cell populations obtained after stimulation of PBMC with different influenza A viruses. Based on the observed recognition patterns of the clonal and polyclonal T cell populations and serology, it is hypothesized that consecutive infections with influenza viruses containing different variants of the epitope select for cross-reactive T cells in vivo.

Mutations, drift, and the influenza archipelago.

Extract: Annual influenza (flu) epidemics in humans affect 5-15% of the population, causing an estimated half million deaths worldwide per year. Antibodies against the viral surface glycoprotein hemagglutinin (HA) provide protective immunity to influenza virus infection and this protein is therefore the primary component of influenza vaccines. However, the antigenic structure of HA has changed significantly over time, a process known as antigenic drift. In as many years, antigenic drift necessitates an update of the influenza vaccine to ensure sufficient efficacy against newly emerging virus variants. Antigenic drift is therefore both the root cause of the enormous public health burden of influenza epidemics, and a primary reason why the virus is such a fascinating pathogen from a scientific perspective. Thousands of influenza viruses are isolated and analyzed each year by the national and international laboratories that form the World Health Organization (WHO) global influenza surveillance network. This worldwide surveillance effort produces the data for the twice-yearly vaccine strain selection meetings, and has resulted in the establishment of a remarkable historical record of the global evolution of this important pathogen. The degree to which immunity induced by one strain is effective against another is mostly dependent on the extent of the antigenic difference between the strains. The analysis of antigenic differences between strains is therefore critical for surveillance and vaccine strain selection, and is also a cornerstone of basic and applied research in virology.

Probabilistic code for DNA recognition by proteins of the EGR family.

A recognition code for protein-DNA interactions would allow for the prediction of binding sites based on protein sequence, and the identification of binding proteins for specific DNA targets. Crystallographic studies of protein-DNA complexes showed that a simple, deterministic recognition code does not exist. Here, we present a probabilistic recognition code (P-code) that assigns energies to all possible base-pair-amino acid interactions for the early growth response factor (EGR) family of zinc-finger transcription factors. The specific energy values are determined by a maximum likelihood method using examples from in vitro randomisation experiments (namely, SELEX and phage display) reported in the literature. The accuracy of the model is tested in several ways, including the ability to predict in vivo binding sites of EGR proteins and other non-EGR zinc-finger proteins, and the correlation between predicted and measured binding affinities of various EGR proteins to several different DNA sites. We also show that this model improves significantly upon the prediction capabilities of previous qualitative and quantitative models. The probabilistic code we develop uses information about the interacting positions between the protein and DNA, but we show that such information is not necessary, although it reduces the number of parameters to be determined. We also employ the assumption that the total binding energy is the sum of the energies of the individual contacts, but we describe how that assumption can be relaxed at the cost of additional parameters.

Is there a code for protein-DNA recognition? Probab(ilistical)ly. . .

Transcriptional regulation of all genes is initiated by the specific binding of regulatory proteins called transcription factors to specific sites on DNA called promoter regions. Transcription factors employ a variety of mechanisms to recognise their DNA target sites. In the last few decades, attempts have been made to describe these mechanisms by general sets of rules and associated models. We give an overview of these models, starting with a historical review of the somewhat controversial issue of a "recognition code" governing protein-DNA interaction. We then present a probabilistic framework in which advantages and disadvantages of various models can be discussed. Finally, we conclude that simplifying assumptions about additivity of interactions are sufficiently justified in many situations (and can be suitably extended in other situations) to allow a unifying concept of a "probabilistic code" for protein-DNA recognition to be defined.