Establishment of a consensus protocol to explore the brain pathobiome in patients with mild cognitive impairment and Alzheimer's disease: Research outline and call for collaboration.

Microbial infections of the brain can lead to dementia, and for many decades microbial infections have been implicated in Alzheimer's disease (AD) pathology. However, a causal role for infection in AD remains contentious, and the lack of standardized detection methodologies has led to inconsistent detection/identification of microbes in AD brains. There is a need for a consensus methodology; the Alzheimer's Pathobiome Initiative aims to perform comparative molecular analyses of microbes in post mortem brains versus cerebrospinal fluid, blood, olfactory neuroepithelium, oral/nasopharyngeal tissue, bronchoalveolar, urinary, and gut/stool samples. Diverse extraction methodologies, polymerase chain reaction and sequencing techniques, and bioinformatic tools will be evaluated, in addition to direct microbial culture and metabolomic techniques. The goal is to provide a roadmap for detecting infectious agents in patients with mild cognitive impairment or AD. Positive findings would then prompt tailoring of antimicrobial treatments that might attenuate or remit mounting clinical deficits in a subset of patients.

Topical application of Porphyromonas gingivalis into the gingival pocket in mice leads to chronic­active infection, periodontitis and systemic inflammation.

Porphyromonas gingivalis (Pg), one of the 'red­complex' perio­pathogens known to play a critical role in the development of periodontitis, has been used in various animal models to mimic human bacteria­induced periodontitis. In order to achieve a more realistic animal model of human Pg infection, the present study investigated whether repeated small­volume topical applications of Pg directly into the gingival pocket can induce local infection, including periodontitis and systemic vascular inflammation in wild­type mice. Freshly cultured Pg was topically applied directly into the gingival pocket of the second molars for 5 weeks (3 times/week). After the final application, the mice were left in cages for 4 or 8 weeks and sacrificed. The status of Pg colony formation in the pocket, gingival inflammation, alveolar bone loss, the expression levels of pro­inflammatory cytokines in the serum and aorta, the presence of anti­Pg lipopolysaccharide (LPS) and gingipain (Kpg and RgpB) antibodies in the serum, as well as the accumulation of Pg LPS and gingipain aggregates in the gingiva and arterial wall were evaluated. The topical application of Pg into the gingival pocket induced the following local and systemic pathohistological changes in mice when examined at 4 or 8 weeks after the final topical Pg application: Pg colonization in the majority of gingival pockets; increased gingival pocket depths; gingival inflammation indicated by the increased expression of TNF­α, IL­6 and IL­1ß; significant loss of alveolar bone at the sites of topical Pg application; and increased levels of pro­inflammatory cytokines, such as TNF­α, IL­1ß, IL­17, IL­13, KC and IFN­Î³ in the serum in comparison to those from mice receiving PBS. In addition, the Pg application/colonization model induced anti­Pg LPS and gingipain antibodies in serum, as well as the accumulation of Pg LPS and gingipain aggregates in the gingivae and arterial walls. To the best of our knowledge, this mouse model represents the first example of creating a more sustained local infection in the gingival tissues of wild­type mice and may prove to be useful for the investigation of the more natural and complete pathogenesis of the bacteria in the development of local oral and systemic diseases, such as atherosclerosis. It may also be useful for the determination of a treatment/prevention/efficacy model associated with Pg­induced colonization periodontitis in mice.

Porphyromonas gingivalis Outer Membrane Vesicles as the Major Driver of and Explanation for Neuropathogenesis, the Cholinergic Hypothesis, Iron Dyshomeostasis, and Salivary Lactoferrin in Alzheimer's Disease.

Porphyromonas gingivalis (Pg) is a primary oral pathogen in the widespread biofilm-induced "chronic" multi-systems inflammatory disease(s) including Alzheimer's disease (AD). It is possibly the only second identified unique example of a biological extremophile in the human body. Having a better understanding of the key microbiological and genetic mechanisms of its pathogenesis and disease induction are central to its future diagnosis, treatment, and possible prevention. The published literature around the role of Pg in AD highlights the bacteria's direct role within the brain to cause disease. The available evidence, although somewhat adopted, does not fully support this as the major process. There are alternative pathogenic/virulence features associated with Pg that have been overlooked and may better explain the pathogenic processes found in the "infection hypothesis" of AD. A better explanation is offered here for the discrepancy in the relatively low amounts of "Pg bacteria" residing in the brain compared to the rather florid amounts and broad distribution of one or more of its major bacterial protein toxins. Related to this, the "Gingipains Hypothesis", AD-related iron dyshomeostasis, and the early reduced salivary lactoferrin, along with the resurrection of the Cholinergic Hypothesis may now be integrated into one working model. The current paper suggests the highly evolved and developed Type IX secretory cargo system of Pg producing outer membrane vesicles may better explain the observed diseases. Thus it is hoped this paper can provide a unifying model for the sporadic form of AD and guide the direction of research, treatment, and possible prevention.

What role do periodontal pathogens play in osteoarthritis and periprosthetic joint infections of the knee?

Through the use of polymerase chain reaction (PCR)-electron spray ionization (ESI)-time of flight (TOF)-mass spectrometry (MS), we identified multiple periodontal pathogens within joint tissues of individuals undergoing replacement arthroplasties of the knee. The most prevalent of the periodontal pathogens were Treponema denticola and Enterococcus faecalis, the latter of which is commonly associated with apical periodontitis. These findings were unique to periprosthetic joint infections (PJI) of the knee and were never observed for PJIs of other lower extremity joints (hip and ankle) or upper extremity joints (shoulder and elbow). These data were confirmed by multiple independent methodologies including fluorescent in situ hybridization (FISH) which showed the bacteria deeply penetrated inside the diseased tissues, and 454-based deep 16S rDNA sequencing. The site-specificity, the tissue investment, and the identical findings by multiple nucleic-acid-based techniques strongly suggests the presence of infecting bacteria within these diseased anatomic sites. Subsequently, as part of a control program using PCR-ESI-TOF-MS, we again detected these same periodontal pathogens in aspirates from patients with osteoarthritis who were undergoing primary arthroplasty of the knee and thus who had no history of orthopedic implants. This latter finding raises the question of whether hematogenic spread of periodontal pathogens to the knee play a primary or secondary-exacerbatory role in osteoarthritis.

Antibody neutralization of retargeted measles viruses.

The measles virus (MV) vaccine lineage is a promising oncolytic but prior exposure to the measles vaccine or wild-type MV strains limits treatment utility due to the presence of anti-measles antibodies. MV entry can be redirected by displaying a polypeptide ligand on the Hemagglutinin (H) C-terminus. We hypothesized that retargeted MV would escape neutralization by monoclonal antibodies (mAbs) recognizing the H receptor-binding surface and be less susceptible to neutralization by human antisera. Using chimeric H proteins, with and without mutations that ablate MV receptor binding, we show that retargeted MVs escape mAbs that target the H receptor-binding surface by virtue of mutations that ablate infection via SLAM and CD46. However, C-terminally displayed domains do not mediate virus entry in the presence of human antibodies that bind to the underlying H domain. In conclusion, utility of retargeted oncolytic measles viruses does not extend to evasion of human serum neutralization.

Epitope dampening monotypic measles virus hemagglutinin glycoprotein results in resistance to cocktail of monoclonal antibodies.

The measles virus (MV) is serologically monotypic. Life-long immunity is conferred by a single attack of measles or following vaccination with the MV vaccine. This is contrary to viruses such as influenza, which readily develop resistance to the immune system and recur. A better understanding of factors that restrain MV to one serotype may allow us to predict if MV will remain monotypic in the future and influence the design of novel MV vaccines and therapeutics. MV hemagglutinin (H) glycoprotein, binds to cellular receptors and subsequently triggers the fusion (F) glycoprotein to fuse the virus into the cell. H is also the major target for neutralizing antibodies. To explore if MV remains monotypic due to a lack of plasticity of the H glycoprotein, we used the technology of Immune Dampening to generate viruses with rationally designed N-linked glycosylation sites and mutations in different epitopes and screened for viruses that escaped monoclonal antibodies (mAbs). We then combined rationally designed mutations with naturally selected mutations to generate a virus resistant to a cocktail of neutralizing mAbs targeting four different epitopes simultaneously. Two epitopes were protected by engineered N-linked glycosylations and two epitopes acquired escape mutations via two consecutive rounds of artificial selection in the presence of mAbs. Three of these epitopes were targeted by mAbs known to interfere with receptor binding. Results demonstrate that, within the epitopes analyzed, H can tolerate mutations in different residues and additional N-linked glycosylations to escape mAbs. Understanding the degree of change that H can tolerate is important as we follow its evolution in a host whose immunity is vaccine induced by genotype A strains instead of multiple genetically distinct wild-type MVs.

Antibody repertoire development in fetal and neonatal piglets XXI. Usage of most VH genes remains constant during fetal and postnatal development.

Usage of variable region gene segments during development of the antibody repertoire in mammals is unresolved in part because of the complexity of the locus in mice and humans and the difficulty of distinguishing intrinsic from extrinsic influences in these species. We present the first vertical studies on VH usage that spans the fetal and neonatal period using the piglet model. We tracked VH usage in DNA rearrangements and in VDJ transcripts throughout 75 days of gestation (DG) in outbred fetuses, thereafter in outbred germfree and colonized isolator piglets, isolator piglets infected with swine influenza and in conventionally reared nematode-infected adults. Seven VH genes account for >90% of the pre-immune repertoire which is the same among tissues and in both transcripts and DNA rearrangements. Statistical modeling supports the view that proportional usage of the major genes remains constant during fetal life and that postnatal usage ranking is similar to that during fetal life. Changes in usage ranking are developmental not antigen dependent. In this species exposure to environmental antigens results in diversification of the repertoire by somatic hypermutation of the same small number of VH genes that comprise the pre-immune repertoire, not by using other VH gene available in the germline. Therefore in swine a small number of VH genes shape the antibody repertoire throughout life questioning the need for extensive VH polygeny.

Evolution of H3N2 influenza virus in a guinea pig model.

Studies of influenza virus evolution under controlled experimental conditions can provide a better understanding of the consequences of evolutionary processes with and without immunological pressure. Characterization of evolved strains assists in the development of predictive algorithms for both the selection of subtypes represented in the seasonal influenza vaccine and the design of novel immune refocused vaccines. To obtain data on the evolution of influenza in a controlled setting, naïve and immunized Guinea pigs were infected with influenza A/Wyoming/2003 (H3N2). Virus progeny from nasal wash samples were assessed for variation in the dominant and other epitopes by sequencing the hemagglutinin (HA) gene to quantify evolutionary changes. Viral RNA from the nasal washes from infection of naïve and immune animals contained 6% and 24.5% HA variant sequences, respectively. Analysis of mutations relative to antigenic epitopes indicated that adaptive immunity played a key role in virus evolution. HA mutations in immunized animals were associated with loss of glycosylation and changes in charge and hydrophobicity in and near residues within known epitopes. Four regions of HA-1 (75-85, 125-135, 165-170, 225-230) contained residues of highest variability. These sites are adjacent to or within known epitopes and appear to play an important role in antigenic variation. Recognition of the role of these sites during evolution will lead to a better understanding of the nature of evolution which help in the prediction of future strains for selection of seasonal vaccines and the design of novel vaccines intended to stimulated broadened cross-reactive protection to conserved sites outside of dominant epitopes.

Selective pressure to increase charge in immunodominant epitopes of the H3 hemagglutinin influenza protein.

The evolutionary speed and the consequent immune escape of H3N2 influenza A virus make it an interesting evolutionary system. Charged amino acid residues are often significant contributors to the free energy of binding for protein-protein interactions, including antibody-antigen binding and ligand-receptor binding. We used Markov chain theory and maximum likelihood estimation to model the evolution of the number of charged amino acids on the dominant epitope in the hemagglutinin protein of circulating H3N2 virus strains. The number of charged amino acids increased in the dominant epitope B of the H3N2 virus since introduction in humans in 1968. When epitope A became dominant in 1989, the number of charged amino acids increased in epitope A and decreased in epitope B. Interestingly, the number of charged residues in the dominant epitope of the dominant circulating strain is never fewer than that in the vaccine strain. We propose these results indicate selective pressure for charged amino acids that increase the affinity of the virus epitope for water and decrease the affinity for host antibodies. The standard PAM model of generic protein evolution is unable to capture these trends. The reduced alphabet Markov model (RAMM) model we introduce captures the increased selective pressure for charged amino acids in the dominant epitope of hemagglutinin of H3N2 influenza (R (2) > 0.98 between 1968 and 1988). The RAMM model calibrated to historical H3N2 influenza virus evolution in humans fit well to the H3N2/Wyoming virus evolution data from Guinea pig animal model studies.

Serological characterization of guinea pigs infected with H3N2 human influenza or immunized with hemagglutinin protein.

BACKGROUND: Recent and previous studies have shown that guinea pigs can be infected with, and transmit, human influenza viruses. Therefore guinea pig may be a useful animal model for better understanding influenza infection and assessing vaccine strategies. To more fully characterize the model, antibody responses following either infection/re-infection with human influenza A/Wyoming/03/2003 H3N2 or immunization with its homologous recombinant hemagglutinin (HA) protein were studied. RESULTS: Serological samples were collected and tested for anti-HA immunoglobulin by ELISA, antiviral antibodies by hemagglutination inhibition (HI), and recognition of linear epitopes by peptide scanning (PepScan). Animals inoculated with infectious virus demonstrated pronounced viral replication and subsequent serological conversion. Animals either immunized with the homologous HA antigen or infected, showed a relatively rapid rise in antibody titers to the HA glycoprotein in ELISA assays. Antiviral antibodies, measured by HI assay, were detectable after the second inoculation. PepScan data identified both previously recognized and newly defined linear epitopes. CONCLUSIONS: Infection and/or recombinant HA immunization of guinea pigs with H3N2 Wyoming influenza virus resulted in a relatively rapid production of viral-specific antibody thus demonstrating the strong immunogenicity of the major viral structural proteins in this animal model for influenza infection. The sensitivity of the immune response supports the utility of the guinea pig as a useful animal model of influenza infection and immunization.

Deceptive imprinting and immune refocusing in vaccine design.

A large number of the world's most widespread and problematic pathogens evade host immune responses by inducing strain-specific immunity to immunodominant epitopes with high mutation rates capable of altering antigenic profiles. The immune system appears to be decoyed into reacting to these immunodominant epitopes that offer little cross protection between serotypes or subtypes. For example, during HIV-1 infection, the immune system reacts strongly to the V1, V2, and/or V3 loops of the surface envelope glycoprotein but not to epitopes that afford broad protection against strain variants. Similarly, the host mounts strain-specific immunity to immunodominant epitopes of the influenza hemagglutinin (HA) protein. A large number of pathogens appear to exploit this weakness in the host immune system by focusing antigenic attention upon highly variable epitopes while avoiding surveillance toward more highly conserved receptor binding sites or other essential functional domains. Because the propensity of the immune system to react against immunodominant strain-specific epitopes appears to be genetically hard-wired, the phenomenon has been termed "deceptive imprinting." In this review, the authors describe observations related to deceptive imprinting in multiple systems and propose strategies for overcoming this phenomenon in the design of vaccines capable of inducing protection against highly variable pathogens.

Perspectives on advancing preventative medicine through vaccinology at the comparative veterinary, human and conservation medicine interface: not missing the opportunities.

Vaccination has historically and remains one of the most cost-effective and safest forms of medicine today. Along with basic understanding of germ theory and sanitation, vaccination, over the past 50 years, has transformed lives and economies in both rich and poor countries by its direct impact on human and animal life--resulting in the eradication of small pox, huge reductions in the burden of previously common human and animal diseases such as polio, typhoid, measles in human medicine and contagious bovine pleuropneumonia, foot-and-mouth disease, screwworm and hog cholera and the verge of eradicating brucellosis, tuberculosis, and pseudorabies in veterinary medicine. In addition vaccination along with other animal production changes has provided the ability to produce otherwise unaffordable animal protein and animal health worldwide. The landscape however on which vaccinology was discovered and applied over the past 200 years, even in the past 10 years has and is undergoing continuous change. For vaccination as a public health tool to have its greatest impacts in human and veterinary medicine, these great medical sciences will have to come together, policy-relevant science for sustainable conservation in developing and developed countries needs to become the norm and address poverty (including lack of basic health care) in communities affected by conservation, and to consider costs and benefits (perceived or not) affecting the well-being of all stakeholders, from the local to the multinational. The need to return to and/or develop new education-based models for turning the tide from the heavily return-on-investment therapeutic era of the last century into one where the investment into the preventative sciences and medicine lead to sustainable cultural and cost-effective public health and economic changes of the future is never more evident than today. The new complex problems of the new millennium will require new educational models that train para- and professional people for thinking and solving complex inter-related biological, ecological, public-, political/economic problems. The single profession that is best positioned to impact vaccinology is Veterinary Medicine. It's melding with human medicine and their role in future comparative and conservation-based programs will be critical to the successful application of vaccines into the 21st century.

Designing immunogens to elicit broadly neutralizing antibodies to the HIV-1 envelope glycoprotein.

To date HIV-1 vaccines have not been able to elicit potent, long lasting, and broadly neutralizing antibodies to the virus. Our knowledge of HIV envelope glycoprotein (Env) structure/function and the existence of a handful of broadly neutralizing antibodies is guiding rational immunogen design. We review here the potential targets on the HIV Env (the glycan shield, the CD4 binding site, the coreceptor binding site, Env fusion intermediates, and the membrane proximal region) and their associated rational immunogen design strategies. Moreover, we discuss immune dampening and immune refocusing strategies designed to counter immunodominant, decoy responses generated by the virus. In this regard, an immunogen design strategy of "in vitro de-evolution" is presented, which begins to distill the HIV Env to its most critical, core functional domains. While we are beginning to have some understanding as to where we would like out immune system to go, we find that our immune repertoire may actually have limits that preclude successful completion of the task at hand. The repertoire limits appear to be a byproduct of autoantibody tolerance mechanisms and the complex structural requirements for effective, potent broadly neutralizing antibodies. Nevertheless, the hope is that through novel insights and creative solutions that we will be able to design immunogens capable of eliciting broadly neutralizing antibodies to the HIV envelope glycoprotein.

Advances in immunology and vaccine discovery report of the United States-European Commission workshop.

As the 21st century unfolds, infectious diseases remain one of the most significant threats to our economy, our food animal production systems, animal welfare, and most importantly, the lives of people worldwide, regardless of their economic standing. The potential use of biological threat agents for terrorism or biowarfare further undermines the security of our society. Arguably, vaccines represent the single most cost-effective, medically delivered strategy for confronting these challenges. The workshop "Advances in Immunology and Vaccine Discovery" was organized to address these challenges, based on the conviction that the interface between immunology and vaccinology offers the best prospects for major breakthroughs in vaccine discovery and development. Six focus areas were identified by workshop organizers: (1) pathogen immune evasion; (2) innate immunity; (3) mucosal immunity; (4) immunogenetics; (5) comparative immunology; and (6) genomics. These areas provided opportunities to elucidate how protective immunity may relate to the disruption of the molecular mechanisms that underlie host-pathogen interactions. A report generated by workshop organizers and participants provides key recommendations and identifies important research gaps, needs, future steps, and potential strategic US-EU collaborations. The report is available on line through ScienceDirect (URL).

HIV-1: the confounding variables of virus neutralization.

The development of an effective vaccine against HIV-1 would be greatly facilitated by the ability to elicit potent, high affinity antibodies that are capable of broad neutralization, viral inactivation and protection against infection and/or disease. New insights into the structure and function of the HIV-1 envelope glycoprotein (Env) that mediates viral fusion and entry may ultimately lead to strategies successful in eliciting these protective antibody responses. Insights have been gained regarding HIV-1 Env attachment and receptor engagement, the fusion process and kinetics, and the structural/functional attributes of Env that allow humoral immune evasion. In addition, studies of a limited number of broadly neutralizing human monoclonal antibodies have shed some light as to how antibodies may penetrate the immune evading armor that HIV-1 has evolved. As the elusive goal of generating these types of antibodies emerge and are developed in the context of generating new candidate HIV-1 vaccines, a relevant in vitro measurement of neutralization by these types of antibodies becomes a complex task. This is in part due to a list of confounding variables which include: the physical and genomic nature (amino acid variation) of the infecting virion, the type of target cells, the concentration and clonality of the reactants, assay format and design, the affinity and kinetics of the reaction, receptors/coreceptors and attachment factors, and soluble host factors. This review will focus on the past, current, and future knowledge required to advance the field of HIV-1 humoral immunity as it impacts future HIV-1 vaccine development.