Luis P. Villarreal

Cutting Edge: Long-Term B Cell Memory in Humans after Smallpox Vaccination

Memory B cells are a central component of humoral immunity, and yet little is known about their longevity in humans. Immune memory after smallpox vaccination (DryVax) is a valuable benchmark for understanding the longevity of B cell memory in the absence of re-exposure to Ag. In this study, we demonstrate that smallpox vaccine-specific memory B cells last for >50 years in immunized individuals. Virus-specific memory B cells initially declined postimmunization, but then reached a plateau ∼10-fold lower than peak and were stably maintained for >50 years after vaccination at a frequency of ∼0.1% of total circulating IgG+ B cells. These persisting memory B cells were functional and able to mount a robust anamnestic Ab response upon revaccination. Additionally, virus-specific CD4+ T cells were detected decades after vaccination. These data show that immunological memory to DryVax vaccine is long-lived and may contribute to protection against smallpox.

Viruses and the evolution of life.

Viral Studies conducted in the 1950s contributed tremendously to the scientific understanding of molecular mechanisms present in living organisms. Viruses and the Evolution of Life is an intriguing presentation of the virus-host relationship, as revealed through an examination of host evolution. This new volume avails the informed reader of a new perspective on the evolution of life while targeting the expert reader with discussion of specific scientific literature. While it is generally accepted among evolutionary biologists that viruses are not self-sustaining and therefore cannot be alive, their impact on life is dramatic. Starting with prebiotic precursors of life, the book features an engaging discussion of various life forms and how each group has been affected by viruses. The author develops the concept of and distinction between acute and persisting viruses, an area of study that has heretofore largely been ignored. The relationship between the familiar disease-causing viruses and those viruses from which they emerge is also clarified. Electronic Only, 395 pages, illustrations, index.

A Hypothesis for DNA Viruses as the Origin of Eukaryotic Replication Proteins

ABSTRACT The eukaryotic replicative DNA polymerases are similar to those of large DNA viruses of eukaryotic and bacterial T4 phages but not to those of eubacteria. We develop and examine the hypothesis that DNA virus replication proteins gave rise to those of eukaryotes during evolution. We chose the DNA polymerase from phycodnavirus (which infects microalgae) as the basis of this analysis, as it represents a virus of a primitive eukaryote. We show that it has significant similarity with replicative DNA polymerases of eukaryotes and certain of their large DNA viruses. Sequence alignment confirms this similarity and establishes the presence of highly conserved domains in the polymerase amino terminus. Subsequent reconstruction of a phylogenetic tree indicates that these algal viral DNA polymerases are near the root of the clade containing all eukaryotic DNA polymerase delta members but that this clade does not contain the polymerases of other DNA viruses. We consider arguments for the polarity of this relationship and present the hypothesis that the replication genes of DNA viruses gave rise to those of eukaryotes and not the reverse direction.

Vaccinia Virus H3L Envelope Protein Is a Major Target of Neutralizing Antibodies in Humans and Elicits Protection against Lethal Challenge in Mice

ABSTRACT The smallpox vaccine is the prototypic vaccine, yet the viral targets critical for vaccine-mediated protection remain unclear in humans. We have produced protein microarrays of a near-complete vaccinia proteome and used them to determine the major antigen specificities of the human humoral immune response to the smallpox vaccine (Dryvax). H3L, an intracellular mature virion envelope protein, was consistently recognized by high-titer antibodies in the majority of human donors, particularly after secondary immunization. We then focused on examining H3L as a valuable human antibody target. Purified human anti-H3L antibodies exhibited substantial vaccinia virus-neutralizing activity in vitro (50% plaque reduction neutralization test [PRNT50] = 44 μg/ml). Mice also make an immunodominant antibody response to H3L after vaccination with vaccinia virus, as determined by vaccinia virus protein microarray. Mice were immunized with recombinant H3L protein to examine H3L-specific antibody responses in greater detail. H3L-immunized mice developed high-titer vaccinia virus-neutralizing antibodies (mean PRNT50 = 1:3,760). Importantly, H3L-immunized mice were subsequently protected against lethal intranasal challenges with 1 or 5 50% lethal doses (LD50) of pathogenic vaccinia virus strain WR, demonstrating the in vivo value of an anti-H3L response. To formally demonstrate that neutralizing anti-H3L antibodies are protective in vivo, we performed anti-H3L serum passive-transfer experiments. Mice receiving H3L-neutralizing antiserum were protected from a lethal challenge with 3 LD50 of vaccinia virus strain WR (5/10 versus 0/10; P < 0.02). Together, these data show that H3L is a major target of the human anti-poxvirus antibody response and is likely to be a key contributor to protection against poxvirus infection and disease.

Viruses are essential agents within the roots and stem of the tree of life.

In contrast with former definitions of life limited to membrane-bound cellular life forms which feed, grow, metabolise and replicate (i) a role of viruses as genetic symbionts, (ii) along with peripheral phenomena such as cryptobiosis and (iii) the horizontal nature of genetic information acquisition and processing broaden our view of the tree of life. Some researchers insist on the traditional textbook conviction of what is part of the community of life. In a recent review [Moreira, D., Lopez-Garcia, P., 2009. Ten reasons to exclude viruses from the tree of life. Nat. Rev. Microbiol. 7, 306-311.] they assemble four main arguments which should exclude viruses from the tree of life because of their inability to self-sustain and self-replicate, their polyphyly, the cellular origin of their cell-like genes and the volatility of their genomes. In this article we will show that these features are not coherent with current knowledge about viruses but that viral agents play key roles within the roots and stem of the tree of life.

Parvovirus variation for disease: a difference with RNA viruses?

The Parvoviridae, a family of viruses with single-stranded DNA genomes widely spread from invertebrates to mammal and human hosts, display a remarkable evolutionary capacity uncommon in DNA genomes. Parvovirus populations show high genetic heterogeneity and large population sizes resembling the quasispecies found in RNA viruses. These viruses multiply in proliferating cells, causing acute, persistent or latent infections relying in the immunocompetence and developmental stage of the hosts. Some parvovirus populations in natural settings, such as carnivore autonomous parvoviruses or primate adeno associated virus, show a high degree of genetic heterogeneity. However, other parvoviruses such as the pathogenic B19 human erythrovirus or the porcine parvovirus, show little genetic variation, indicating different virus-host relationships. The Parvoviridae evolutionary potential in mammal infections has been modeled in the experimental system formed by the immunodeficient scid mouse infected by the minute virus of mice (MVM) under distinct immune and adaptive pressures. The sequence of viral genomes (close to 10(5) nucleotides) in emerging MVM pathogenic populations present in the organs of 26 mice showed consensus sequences not representing the complex distribution of viral clones and a high genetic heterogeneity (average mutation frequency 8.3 x 10(-4) substitutions/nt accumulated over 2-3 months). Specific amino acid changes, selected at a rate up to 1% in the capsid and in the NS2 nonstructural protein, endowed these viruses with new tropism and increased fitness. Further molecular analysis supported the notion that, in addition to immune pressures, the affinity of molecular interactions with cellular targets, as the Crml nuclear export receptor or the primary capsid receptor, as well as the adaptation to tissues enriched in proliferating cells, are major selective factors in the rapid parvovirus evolutionary dynamics.

Natural Biology of Polyomavirus Middle T Antigen

SUMMARY “It has been commented by someone that ‘polyoma’ is an adjective composed of a prefix and suffix, with no root between—a meatless linguistic sandwich” (C. J. Dawe). The very name “polyomavirus” is a vague mantel: a name given before our understanding of these viral agents was clear but implying a clear tumor life-style, as noted by the late C. J. Dawe. However, polyomavirus are not by nature tumor-inducing agents. Since it is the purpose of this review to consider the natural function of middle T antigen (MT), encoded by one of the seemingly crucial transforming genes of polyomavirus, we will reconsider and redefine the virus and its MT gene in the context of its natural biology and function. This review was motivated by our recent in vivo analysis of MT function. Using intranasal inoculation of adult SCID mice, we have shown that polyomavirus can replicate with an MT lacking all functions associated with transformation to similar levels to wild-type virus. These observations, along with an almost indistinguishable replication of all MT mutants with respect to wild-type viruses in adult competent mice, illustrate that MT can have a play subtle role in acute replication and persistence. The most notable effect of MT mutants was in infections of newborns, indicating that polyomavirus may be highly adapted to replication in newborn lungs. It is from this context that our current understanding of this well-studied virus and gene is presented.

Viral ancestors of antiviral systems.

All life must survive their corresponding viruses. Thus antiviral systems are essential in all living organisms. Remnants of virus derived information are also found in all life forms but have historically been considered mostly as junk DNA. However, such virus derived information can strongly affect host susceptibility to viruses. In this review, I evaluate the role viruses have had in the origin and evolution of host antiviral systems. From Archaea through bacteria and from simple to complex eukaryotes I trace the viral components that became essential elements of antiviral immunity. I conclude with a reexamination of the ‘Big Bang’ theory for the emergence of the adaptive immune system in vertebrates by horizontal transfer and note how viruses could have and did provide crucial and coordinated features.

The Source of Self: Genetic Parasites and the Origin of Adaptive Immunity

Stable colonization of the host by viruses (genetic parasites) can alter the systems of host identity and provide immunity against related viruses. To attain the needed stability, some viruses of prokaryotes (P1 phage) use a strategy called an addiction module. The linked protective and destructive gene functions of an addiction module insures both virus persistence but will also destroy cells that interrupt this module and thereby prevent infection by competitors. Previously, I have generalized this concept to also include persistent and lytic states of virus infection, which can be considered as a virus addiction module. 1 Such states often involve defective viruses. In this report, I examine the origin of the adaptive immune system from the perspective of a virus addiction module. The likely role of both endogenous and exogenous retroviruses, DNA viruses, and their defective elements is considered in the origin of all the basal components of adaptive immunity (T‐cell receptor, RAG‐mediated gene rearrangement, clonal lymphocyte proliferation, antigen surface presentation, apoptosis, and education of immune cells). It is concluded that colonization by viruses and their defectives provides a more coherent explanation for the origin of adaptive immunity.

How viruses shape the tree of life

Consideration of virus evolution only from a disease perspective has provided a limited view of virus–host evolution. Such views assume all viruses fit predator/prey models for replication, but fail to explain the origin of disease or how viruses might make significant contributions to host evolution. On a long evolutionary time scale, the ability of a virus to persist in an individual host is a much more prevalent life strategy. Persistence can explain both origins of most viral disease and virus–host evolutionary stability. However, persistence (both genomic and epigenomic) is a much more complex and demanding virus–host relationship that is difficult to study. We must change our attitudes towards persistence if we are to understand how viruses shape the tree of life. Readers of virus evolution literature are most likely to be interested in understanding viral disease and its origins. How can we understand the seemingly endless reports of new or re-emerging viral threats to human and animal health? How do these agents evolve, where do they come from, and how does this affect host evolution? Serious disease demands serious attention, so we have focused our efforts to understand the relevant relationships. Progress has been good and, although Darwinian concepts clearly apply to viruses, additional concepts such as clonal generation of quasispecies and error catastrophe, have expanded our understanding of virus evolution and led us to believe that no virus genomes exist as a single nucleotide sequence as they spread rapidly into a mutant spectra. Population selection, population memory, mutant clouds, Shannon entropy and Hamming distance are all concepts that appear to be consistent with the experimental study of viral