Structural basis for llama nanobody recognition and neutralization of HIV-1 at the CD4-binding site.

Nanobodies can achieve remarkable neutralization of genetically diverse pathogens, including HIV-1. To gain insight into their recognition, we determined crystal structures of four llama nanobodies (J3, A12, C8, and D7), all of which targeted the CD4-binding site, in complex with the HIV-1 envelope (Env) gp120 core, and determined a cryoelectron microscopy (cryo-EM) structure of J3 with the Env trimer. Crystal and cryo-EM structures of J3 complexes revealed this nanobody to mimic binding to the prefusion-closed trimer for the primary site of CD4 recognition as well as a secondary quaternary site. In contrast, crystal structures of A12, C8, and D7 with gp120 revealed epitopes that included portions of the gp120 inner domain, inaccessible on the prefusion-closed trimer. Overall, these structures explain the broad and potent neutralization of J3 and limited neutralization of A12, C8, and D7, which utilized binding modes incompatible with the neutralization-targeted prefusion-closed conformation of Env.

Luc Montagnier (1932-2022).

Discoverer of the human immunodeficiency virus.

Emergence of epidemic diseases: zoonoses and other origins.

Infectious diseases emerge via many routes and may need to overcome stepwise bottlenecks to burgeon into epidemics and pandemics. About 60% of human infections have animal origins, whereas 40% either co-evolved with humans or emerged from non-zoonotic environmental sources. Although the dynamic interaction between wildlife, domestic animals, and humans is important for the surveillance of zoonotic potential, exotic origins tend to be overemphasized since many zoonoses come from anthropophilic wild species (for example, rats and bats). We examine the equivocal evidence of whether the appearance of novel infections is accelerating and relate technological developments to the risk of novel disease outbreaks. Then we briefly compare selected epidemics, ancient and modern, from the Plague of Athens to COVID-19.

A perspective on the early days of RAS research.

The name of the oncogene, ras, has its origin in studies of murine leukemia viruses in the 1960s by Jenny Harvey (H-ras) and by Werner Kirsten (K-ras) which, at high doses, produced sarcomas in rats. Transforming retroviruses were isolated, and its oncogene was named ras after rat sarcoma. From 1979, cellular ras sequences with transforming properties were identified by transfection of tumor DNA initially by Robert Weinberg from rodent tumors, and the isolation of homologous oncogenes from human tumors soon followed, including HRAS and KRAS, and a new member of the family named NRAS. I review these discoveries, placing emphasis on the pioneering research of Christopher Marshall and Alan Hall, who subsequently made immense contributions to our understanding of the functions of RAS and related small GTPases to signal transduction pathways, cell structure, and the behavior of normal and malignant cells.

Super Potent Bispecific Llama VHH Antibodies Neutralize HIV via a Combination of gp41 and gp120 Epitopes.

Broad and potent neutralizing llama single domain antibodies (VHH) against HIV-1 targeting the CD4 binding site (CD4bs) have previously been isolated upon llama immunization. Here we describe the epitopes of three additional VHH groups selected from phage libraries. The 2E7 group binds to a new linear epitope in the first heptad repeat of gp41 that is only exposed in the fusion-intermediate conformation. The 1B5 group competes with co-receptor binding and the 1F10 group interacts with the crown of the gp120 V3 loop, occluded in native Env. We present biophysical and structural details on the 2E7 interaction with gp41. In order to further increase breadth and potency, we constructed bi-specific VHH. The combination of CD4bs VHH (J3/3E3) with 2E7 group VHH enhanced strain-specific neutralization with potencies up to 1400-fold higher than the mixture of the individual VHHs. Thus, these new bivalent VHH are potent new tools to develop therapeutic approaches or microbicide intervention.

Nanobodies that Neutralize HIV.

Nanobodies or VHH (variable domains of heavy-chain only antibodies) are derived from camelid species such as llamas and camels. Nanobodies isolated and selected through phage display can neutralize a broad range of human immunodeficiency virus type 1 (HIV-1) strains. Nanobodies fit into canyons on the HIV envelope that may not be accessible to IgG (immunoglobulin G) containing both heavy and light chains, and they tend to have long CDR3 (complementarity-determining region 3) loops that further enhance recognition of otherwise cryptic epitopes. Nanobodies are readily expressed at high levels in bacteria and yeast, as well as by viral vectors, and they form relatively stable, heat-resistant molecules. Nanobodies can be linked to human Fc chains to gain immune effector functions. Bivalent and trivalent nanobodies recognizing the same or distinct epitopes on the envelope glycoproteins, gp120 and gp41, greatly increase the potency of HIV-1 neutralization. Nanobodies have potential applications for HIV-1 diagnostics, vaccine design, microbicides, immunoprophylaxis, and immunotherapy.

Open questions: knowing who's who in multicellular animals is not always as simple as we imagine.

The ability of certain tumor cells of mammals and molluscs to spread from the original host to others reopens the question of distinguishing self from non-self. It is part of a wider phenomenon of cellular parasitism and cell chimerism including germ cells.

Remembering Jan Svoboda: A Personal Reflection.

The Czech scientist Jan Svoboda was a pioneer of Rous sarcoma virus (RSV). In the 1960s, before the discovery of reverse transcriptase, he demonstrated the long-term persistence of the viral genome in non-productive mammalian cells, and he supported the DNA provirus hypothesis of Howard Temin. He showed how the virus can be rescued in the infectious form and elucidated the replication-competent nature of the Prague strain of RSV later used for the identification of the src oncogene. His studies straddled molecular oncology and virology, and he remained an active contributor to the field until his death last year. Throughout the 50 years that I was privileged to know Svoboda as my mentor and friend, I admired his depth of scientific inquiry and his steadfast integrity in the face of political oppression.

Molecular Signatures of Regression of the Canine Transmissible Venereal Tumor.

The canine transmissible venereal tumor (CTVT) is a clonally transmissible cancer that regresses spontaneously or after treatment with vincristine, but we know little about the regression mechanisms. We performed global transcriptional, methylation, and functional pathway analyses on serial biopsies of vincristine-treated CTVTs and found that regression occurs in sequential steps; activation of the innate immune system and host epithelial tissue remodeling followed by immune infiltration of the tumor, arrest in the cell cycle, and repair of tissue damage. We identified CCL5 as a possible driver of CTVT regression. Changes in gene expression are associated with methylation changes at specific intragenic sites. Our results underscore the critical role of host innate immunity in triggering cancer regression.

Human oncogenic viruses: nature and discovery.

Seven kinds of virus collectively comprise an important cause of cancer, particularly in less developed countries and for people with damaged immune systems. Discovered over the past 54 years, most of these viruses are common infections of humankind for which malignancy is a rare consequence. Various cofactors affect the complex interaction between virus and host and the likelihood of cancer emerging. Although individual human tumour viruses exert their malignant effects in different ways, there are common features that illuminate mechanisms of oncogenesis more generally, whether or not there is a viral aetiology.This article is part of the themed issue 'Human oncogenic viruses'.

Towards an ultra-rapid smartphone- connected test for infectious diseases.

The development is reported of an ultra-rapid, point-of-care diagnostic device which harnesses surface acoustic wave (SAW) biochips, to detect HIV in a finger prick of blood within 10 seconds (sample-in-result-out). The disposable quartz biochip, based on microelectronic components found in every consumer smartphone, is extremely fast because no complex labelling, amplification or wash steps are needed. A pocket-sized control box reads out the SAW signal and displays results electronically. High analytical sensitivity and specificity are found with model and real patient blood samples. The findings presented here open up the potential of consumer electronics to cut lengthy test waiting times, giving patients on the spot access to potentially life-saving treatment and supporting more timely public health interventions to prevent disease transmission.

Unravelling the Molecular Basis of High Affinity Nanobodies against HIV p24: In Vitro Functional, Structural, and in Silico Insights.

Preventing the spread of infectious diseases remains an urgent priority worldwide, and this is driving the development of advanced nanotechnology to diagnose infections at the point of care. Herein, we report the creation of a library of novel nanobody capture ligands to detect p24, one of the earliest markers of HIV infection. We demonstrate that these nanobodies, one tenth the size of conventional antibodies, exhibit high sensitivity and broad specificity to global HIV-1 subtypes. Biophysical characterization indicates strong 690 pM binding constants and fast kinetic on-rates, 1 to 2 orders of magnitude better than monoclonal antibody comparators. A crystal structure of the lead nanobody and p24 was obtained and used alongside molecular dynamics simulations to elucidate the molecular basis of these enhanced performance characteristics. They indicate that binding occurs at C-terminal helices 10 and 11 of p24, a negatively charged region of p24 complemented by the positive surface of the nanobody binding interface involving CDR1, CDR2, and CDR3 loops. Our findings have broad implications on the design of novel antibodies and a wide range of advanced biomedical applications.

Exchange of Genetic Sequences Between Viruses and Hosts.

Although genetic transfer between viruses and vertebrate hosts occurs less frequently than gene flow between bacteriophages and prokaryotes, it is extensive and has affected the evolution of both parties. With retroviruses, the integration of proviral DNA into chromosomal DNA can result in the activation of adjacent host gene expression and in the transduction of host transcripts into retroviral genomes as oncogenes. Yet in contrast to lysogenic phage, there is little evidence that viral oncogenes persist in a chain of natural transmission or that retroviral transduction is a significant driver of the horizontal spread of host genes. Conversely, integration of proviruses into the host germ line has generated endogenous retroviral genomes (ERV) in all vertebrate genomes sequenced to date. Some of these genomes retain potential infectivity and upon reactivation may transmit to other host species. During mammalian evolution, sequences of retroviral origin have been repurposed to serve host functions, such as the viral envelope glycoproteins crucial to the development of the placenta. Beyond retroviruses, DNA viruses with complex genomes have acquired numerous genes of host origin which influence replication, pathogenesis and immune evasion, while host species have accumulated germline sequences of both DNA and RNA viruses. A codicil is added on lateral transmission of cancer cells between hosts and on migration of host mitochondria into cancer cells.

Human endogenous retroviruses: friend or foe?

The integration of proviral DNA into host chromosomal DNA as an obligatory step in the replication cycle of retroviruses is a natural event of genetic recombination between virus and host. When integration occurs in cells of the germ line, it results in mendelian inheritance of viral sequences that we call endogenous retroviruses (ERV) and HERV for humans. HERVs and host often establish a symbiotic relationship, especially in the placenta and in pluripotent embryonic stem cells, but HERVs occasionally have deleterious consequences for the host. This special issue of APMIS features the fascinating relationships between HERV and humans in health and disease.

What's the host and what's the microbe? The Marjory Stephenson Prize Lecture 2015.

The interchange between retroviruses and their hosts is an intimate one because retroviruses integrate proviral DNA into host chromosomal DNA as an obligate step in the replication cycle. This has resulted in the occasional transduction of host genes into retroviral genomes as oncogenes, and also led to the integration of viral genomes into the host germ line that gives rise to endogenous retroviruses. I shall reflect on the evolutionary consequences of these events for virus and host. Then, I shall discuss the emergence of non-viral infections of host origin, namely, how malignant cells can give rise to eukaryotic single cell 'parasites' that colonize new hosts and how these in turn have been colonized by host mitochondria.

Structural Repertoire of HIV-1-Neutralizing Antibodies Targeting the CD4 Supersite in 14 Donors.

The site on the HIV-1 gp120 glycoprotein that binds the CD4 receptor is recognized by broadly reactive antibodies, several of which neutralize over 90% of HIV-1 strains. To understand how antibodies achieve such neutralization, we isolated CD4-binding-site (CD4bs) antibodies and analyzed 16 co-crystal structures -8 determined here- of CD4bs antibodies from 14 donors. The 16 antibodies segregated by recognition mode and developmental ontogeny into two types: CDR H3-dominated and VH-gene-restricted. Both could achieve greater than 80% neutralization breadth, and both could develop in the same donor. Although paratope chemistries differed, all 16 gp120-CD4bs antibody complexes showed geometric similarity, with antibody-neutralization breadth correlating with antibody-angle of approach relative to the most effective antibody of each type. The repertoire for effective recognition of the CD4 supersite thus comprises antibodies with distinct paratopes arrayed about two optimal geometric orientations, one achieved by CDR H3 ontogenies and the other achieved by VH-gene-restricted ontogenies.