EXPERT REVIEW OF https://doi.org/10.1080/14760584.2019.1674144

REVIEW How vaccines work: immune effector mechanisms and designer vaccines Stewart Sell Wadsworth Center, New York State Department of Health, Empire State Plaza, Albany, NY, USA

ABSTRACT ARTICLE HISTORY Introduction: Three major advances have led to increase in length and quality of human life: Received 6 June 2019 increased food production, improved sanitation and induction of specific adaptive immune Accepted 25 September 2019 responses to infectious agents (). Which has had the most impact is subject to debate. KEYWORDS The number and variety of agents and the mechanisms that they have evolved to allow Vaccines; immune effector them to colonize humans remained mysterious and confusing until the last 50 years. Since then mechanisms; science has developed complex and largely successful ways to immunize against many of these neutralization; receptor infections. blockade; anaphylactic Areas covered: Six specific immune defense mechanisms have been identified. neutralization, cytolytic, reactions; - immune complex, anaphylactic, T-cytotoxicity, and delayed hypersensitivity. The role of each of these mediated cytolysis; immune immune effector mechanisms in immune responses induced by vaccination against specific infectious complex reactions; T-cell- mediated cytotoxicity; agents is the subject of this review. delayed hypersensitivity Expertopinion: In the past development of specific vaccines for infections agents was largely by trial and error. With an understanding of the natural history of an and the effective immune response to it, one can select the method of vaccination that will elicit the appropriate immune effector mechanisms (designer vaccines). These may act to prevent infection (prevention) or eliminate an established on ongoing infection (therapeutic). Literature search: The primary literature source is Pub Med. Secondary source is Wikipedia.

1. Introduction receptors (paratopes) on induced immunoglobulin molecules 1.1. Space invaders () or T cells to epitopes (antigens) on individual infectious agents. Thewidevarietyandabilitiesofinfectiousorganismstoevade human defense mechanisms is analogous to the invaders of Earth 1.4. Immune effector mechanisms from Space envisioned in movies and comic books. By ‘clever design’ or evolution, we have developed a variety of both specific Adaptive immunity works by specific immune effector (adaptive) and nonspecific (natural or innate) means to defend mechanisms directed to counteract the properties of infec- against the myriad of invaders of our space, i.e. our bodies [1]. tious agents that allow them to survive, proliferate and Unfortunately for us, either the design is imperfect or evolution cause disease. Prior to the publication of the book ‘Clinical includes not only development of immune protection for us but Aspects of ’ by Phillip Gell and Robin Coombs in also a means for invaders to evade our protective mechanisms. 1963 [4], there was no useful classification of adaptive immune reactions or immune-mediated diseases. There was a long list 1.2. Natural immunity of in vitro reactions, inflammatory phenomena, and diseases that provided a confusing and overwhelming view of immu- Nonspecific innate or natural immunity mechanisms do not require nology and immunity. Gell and Coombs proffered four or specific recognition of invaders. These include immune mechanisms; chemical and physical barriers, natural killer cells, activated macro- Type I: Atopic or anaphylactic; phages, and humoral components such as lysozyme, acute phase Type II: Cytolytic, , complement, natural antibodies, etc [2]. These are not Type III: Immune complex, activated by vaccination but may contribute to a more robust Type IV: Delayed hypersensitivity (DTH). adaptive immune response (danger hypothesis [3]). This has been expanded by 3 [1]: Neutralization added; DTH has been divided into two: DTH and T-cell cytotoxicity (T-CTL), 1.3. Adaptive immunity and Granulomatous reactions added. Examples of the ‘double-edged sword’ of immune effector The specific adaptive immune system includes a variety of mechanisms in protective immunity and in disease are listed immune effector mechanisms directed by specific regions of in Table 1. Immune effector mechanisms may not only act

CONTACT Stewart Sell [email protected] Wadsworth Center, New York State Department of Health, Empire State Plaza, Albany, NY 12201, USA © 2019 Informa UK Limited, trading as Taylor & Francis Group 2 S. SELL

Table 1. The ‘double-edged sword’ of immune effector mechanisms. Immune effector mechanism Protective function Destructive function Neutralization (inactivation) Diphtheria, , cholera Endotoxin neutralization Insulin resistance, bleeding (blood clotting factors (receptor blockade) Block viral-cell interactions Myasthenia gravis, Grave’s disease Cytotoxic Bacteriolysis Anemia, leukopenia Immune complex Acute , opsonization Vasculitis, glomerulonephritis, Arthritis Atopic Vasodilation, intestinal parasites Asthma, anaphylaxis, hay T-cell cytotoxicity Viral exanthems, influenza Contact dermatitis, graft rejection Delayed hypersensitivity Tuberculosis, leprosy Auto-allergies, post-vaccinal Granulomatous Tuberculosis, helminths Sarcoidosis, berylliosis effectively against foreign invaders, but also may cause dis- infection, and if not may lead to irreversible tissue damage ease when not appropriately applied or when directed to self and scarring. Activation of the complement system may be (auto-immunity). The objective of this review is to list the shared by at least three mechanisms: Neutralization: removal specific immune effector mechanisms that are activated and of molecules by opsonization; Cytolysis; lysis of red blood cells, function in defense against infection after vaccination [5]. and Immune Complex: attraction and activation of polymor- phonuclear leukocytes by complement components. 1.4.1. Neutralization reactions The effect of neutralization reactions is to inactivate or block 1.4.4. Atopic and anaphylactic reactions the activity of biologically active molecules. The may be These reactions serve to open blood vessels during inflamma- accomplished by two mechanisms: inactivation or receptor tion and play a general role in protection against infection by blockade. Inactivation by antibody reactions with a biologi- initiating inflammation (vasodilation). For intestinal worm cally active molecule (hormone, enzyme or toxin) usually infestations atopic reactions may cause expulsion of the involves alteration in the tertiary structure so that the biologic worms (Helminths). Anaphylactic reactions in the lung lead agent can no longer function [1]. This is the primary mode of to asthma and in the peripheral blood vessels to generalized action of vaccines against diphtheria, tetanus, pertussis and vasodilation (shock). In viral lung infections sneezing and cholera . Antibodies may also eliminate antigens by coughing may not only result to elimination of virus and activating macrophages to remove them from the circulation virus infected cells, but also contribute to spread of the ‘opsonization’. This is believed to be one mode of action of disease. antibodies to Tau following immunization of individuals with 1.4.5. T-cell cytotoxicity Alzheimer’s disease (see below). Neutralizing antibodies may Cytotoxic T-cells (T-CTLs) react against viral infected cells and also block the interaction between a ligand and receptor so are a mechanism for elimination of virus infections. This is that they cannot bind. To replicate viruses must enter cells especially important in small pox, measles, papilloma virus, and they do this through specific receptor-ligand interactions. hepatitis virus and virus infections as well as influenza, Antibodies to the surface ligands on viruses prevent binding and is the primary immune mechanism in tumor immunity. The to receptors on cells (receptor blockade). Antibodies to recep- classic T-CTL reaction is contact dermatitis (poison ivy) where tors on normal cells are responsible for some diseases, such as specific T-CTL attack a foreign antigen attached to skin epithe- myasthenia gravis, pernicious anemia, hemophilia, and some lial cells. This is like the epithelial reaction seen in small pox and types of diabetes. Auto-antibodies to a receptor on thyroid measles. Autoimmune diseases due to T-CTL are Hashimoto’s cells actually activate thyroid hormone secretion and cause disease (thyroiditis), Chron’s disease, psoriasis, and vitiligo. hyperthyroidism [1]; 1.4.6. Delayed hypersensitivity 1.4.2. Cytotoxic reactions DTH is mediated by the activation of monocytes by products Antibodies to cell surface receptors may activate complement- of CD4 + T cells reacting with antigen. It is the primary mediated lysis. This mechanism usually is directed to cells in mechanism of defense in tuberculosis, leprosy, syphilis, and suspension, such as blood cells. This mechanism is active against candidiasis. Tuberculosis and leprosy infect macrophages. bacterial infections, such as streptococcus, staphylococcus and CD4 + T cells react with antigen on the infected cells, activat- salmonella, and may cause hemolytic anemia, loss of platelets ing phagocytosis and digestion of the organisms. DTH is the (thrombocytopenia) and loss of white blood cells effector mechanism for demyelinating diseases. (agranulocytosis). 1.4.7. Granulomatous reactions 1.4.3. Immune complex reactions Granulomatous reactions may be formed after activation of a Antibody and complement (toxic complex reactions) activate specific or nonspecific mechanism. They are space occupying acute inflammatory reactions seen in many bacterial infections collections of monocytes, giant cells and fibrous tissue that resulting in opsonization of and infiltration and acti- form in response to material that cannot be easily broken vation of polymorphonuclear cells. This mechanism is respon- down and removed by macrophages Granulomas form when sible for abscess formation in bacterial infections DTH is unable to clear an infection. If effective they serve to (Streptococcal, Streptococcal, etc.) and, if effective, cure the wall off infected cells or organisms, such as fungi. If immunity EXPERT REVIEW OF VACCINES 3 is not effective the granulomas continue to grow and produce accomplished by blocking the reaction of the infectious disease by occupying space. This is seen in military tubercu- agent with the cells that it will infect; therapy is directed to losis and in diseases such as berylliosis. Sarcoidosis and rheu- killing already infected cells or organisms that survive outside matoid nodules. For more details on immune effector of cells. mechanisms and how they function in health and disease please see reference [1]. 2. Vaccines and immune effector mechanisms 1.4.8. design 2.1. Toxin neutralization Vaccines should be designed to stimulate the effector mechanism that is known to be effective from the natural 2.1.1. Diphtheria history of each infection [5] and the immune response that is The signs and symptoms of diphtheria are caused by a toxin effective against it. The immune effector mechanisms called released by infections with Corynebacterium diphtheriae. The into play by specific vaccination against the infectious agents bacteria infect the salivary glands in the throat and can cause are listed Table 2. Effective vaccines may act in two ways: difficult breathing and swollen lymph nodes. The toxin gene is induction of an immune mechanism that will prevent infection encoded by a bacteriophage (a virus that infects the bacteria). or one that will provide for therapy once the infection has The toxin enters the cytoplasm of human epithelial cells and occurred. For example, in virus infections prevention is inhibits protein synthesis [6]. The toxin is a single polypeptide

Table 2. Putative preventive and therapeutic adaptive immune responses to specific infections agents induced by vaccination. Mechanism Preventive Therapeutic Toxin neutralization inactivation T-CTL Tetanus Toxin inactivation Toxin inactivation Receptor blockade and toxin neutralization Pertussis IgA receptor blockade Toxin inactivation, T-CTL, DTH Cholera IgA receptor blockade Toxin inactivation IgA receptor blockade Toxin inactivation Receptor blockade and immune complex reaction H. Influenza Receptor blockade Immune complex Meningococcus Receptor blockade Immune complex (opsonization) Pneumococcus Receptor blockade Immune complex Streptococcus A IgA Receptor blockade Immune complex (opsonization) Receptor blockade and T-CTL Viral Exanthems Receptor blockade T-CTL Measles Receptor blockade T-CTL Rubella Receptor blockade T-CTL Varicella Receptor blockade T-CTL Influenza Receptor blockade T-CTL Mumps Receptor blockade T-CTL Ebola Receptor blockade T-CTL Rotavirus Receptor blockade T-CTL HIV Receptor blockade T-CTL Receptor blockade T-CTL Hepatitis Receptor blockade T-CTL Polio Receptor blockade T-CTL Chagas disease Receptor blockade T-CTL Epstein Barr virus Receptor blockade T-CTL, DTH Receptor blockade and DTH Yellow fever Receptor blockade DTH Japanese encephalitis Receptor blockade DTH Dengue Receptor blockade DTH virus Receptor blockade DTH West Nile Receptor blockade DTH Zika Receptor blockade DTH Typhoid fever Receptor blockade DTH Cytolytic Malaria Cytotoxic antibody T-CTL Atopic Helminths Atopic DTH Schistosomiasis Atopic DTH Immune complex Staphylococcus aureus Immune complex Toxin inactivation Immune complex DTH T-CTL Papilloma T-CTL DTH Tuberculosis and leprosy DTH DTH Syphilis DTH DTH Candidiasis DTH DTH 4 S. SELL chain of 535 amino acids consisting of two subunits linked by termini. Since the organisms are in dead tissue and do not infect bridges [7]. One unit ‘B’ binds to the cell surface the body, an immune response to C. tetani itself is not required through epithelial growth factor like receptors and the other for protection [1]. unit ‘A’ penetrates the cytoplasm through endocytosis where it inhibits RNA translation and causes death of laryngeal and 2.2. Receptor blockade and toxin neutralization bronchial cells. Prominent are pseudo-membranes, which are formed from waste products and protein from intoxicated 2.2.1. Pertussis cells. The Schick test developed by Bela Schick in 1925 was Bordetella pertussis is a Gram-negative, aerobic coccobacillus used to test for immunity to the toxin. In this test 0.1 ml of which infects the ciliated epithelium of the larynx and pul- toxin is injected into the skin of one arm, and 0.1 ml of monary bronchi leading to cough and difficult breathing inactivated toxin into the other. Individuals with neutralizing (whooping cough) [16]. During infection it penetrates the antibody will have no reaction at either site; those without epithelium and macrophages through reaction of a filamen- antibody will have acute inflammation at the toxin site but not tous with receptors on cells and multiplies the inactivated toxin site [8]. The vaccine is usually included in within cells where it produces at least four toxins [17]. The trivalent diphtheria, pertussis, tetanus (DPT) . As toxins inflict local mucosal damage with heavy exudates char- with tetanus vaccination, 10-year booster injections are highly acteristic of the infection. This sets the stage for multiple recommended. The is produced by heat treatment or possible immune-protective mechanisms [16]. First innate partial denaturation of toxin released in vitro cultures of C. immune mechanisms (dendritic cells, NK cells, macrophages diphtheriae with formalin [9] and absorption to aluminum and polymorphonuclear cells) may limit the infection, but salts (alum), which increases immunogenicity. The vaccine eventually specific active immunity is much more effective. limits the severity of the disease so that an immune response This is induced by the five-component can eventually eliminate infected cells (T-CTL). Use of the which includes acellular pertussis antigens, detoxified pertus- vaccine has resulted in a more than 90% decrease in the sis toxin (toxoid), filamentous hemagglutinin, pertactin and number of cases globally [10]. fimbriae types 2 and 3, absorbed to alum [18]. Specific active immunity includes antibodies to pertussis toxins, antibody to 2.1.2. Tetanus filamentous hemagglutinin, T-cytotoxic cells and delayed The vaccine for tetanus is an inactivated form of a toxin which hypersensitivity (T-cell-mediated macrophage activation) [16]. causes life-threatening muscle contractions particularly in the Thus, the multi-directed immune response blocks cell entry, jaw and neck muscles (lock Jaw) that interfere with the ability to inhibits toxins, and directs T-cytotoxic cells to infected epithe- breath [11]. Tetanus is caused by extremely durable spores of lial cells and DTH to infected macrophages. The widely used the tetani bacteria that live in soil, saliva and manure pertussis vaccines induce good antibody responses, but weak [12]. The spores enter tissues usually through a cut or wound cellular responses. Recently pertussis vaccines have been mod- and convert to the vegetative form in the anaerobic environ- ified to induce cell-mediated immunity by replacing alum with ment of damaged tissue. The toxins are released from the alternative adjuvants, such as toll-like receptor. It has also damaged tissue into the body. There are two toxins, been suggested that pertussis vaccination might be more and [13]. Tetanolysin damages viable tissue sur- effective if given separately from diphtheria and tetanus rounding the infection and contributes to growth of the vege- immunizations [16]. tative form of the organism. Tetanospasmin is very potent, enters the circulation binds to nerve terminals and produces 2.2.2. Cholera the muscle contractions characteristic of the clinical symptoms. Cholera is an acute diarrhea caused by Gram-negative bacter- In 2015 there were about 209,000 infections and 60,000 deaths ium Vibrio cholerae. Cholera was described in India in the fifth globally; down from 356,000 deaths in 1990 [14]. The tetanus century BC and spread in a world-wide epidemic in 1817. vaccine is made of inactivated tetanospasmin toxin (toxoid) that There have been 7 epidemics since then, the last in 1961. has immunizing epitopes but no biologic activity. The toxoid is The WHO estimates that there are up to 5 million cases per made by treating the toxin with chemicals, such as formalde- year [19]. It is acquired orally from contaminated water and hyde [12]. The amount of antibody produced is measured by can be prevented by appropriate sanitation. In the 1850s, a international units (greater than 0.01 IU) is considered protec- London physician, John Snow, demonstrated that the source tive. The international unit is determined by enzyme-linked of a cholera epidemic was contaminated well water, and was immune-absorbent assay (ELISA) and compared to the World able to halt an epidemic. It is now endemic in many areas of Health Organization (WHO) reference sera. This level is usually Asia and Africa and in the past 10 years devastating epidemics greatly exceeded after the first immunization but decreases of cholera have broken out in Angola, Ethiopia, Zimbabwe, with time, so booster immunizations are recommended every Pakistan, Somalia, Vietnam and Haiti. V. cholerae has more 10 years. The vaccine is essentially 100% effective [12] and than 200 serotypes based on the O almost all cases of tetanus occur in people who are unimmu- Antigen; only types O1 and O 139 cause epidemics. nized or have not had a booster in the preceding 10 years [14]. Originally susceptible to most antibiotics, now antibiotic resis- The tetanus toxoid is usually included in a form called DTaP tant strains are more common. After ingestion most organisms which includes diphtheria and pertussis vaccine [15]. The are killed by gastric acid, but surviving organisms colonize the mechanism of action is neutralization by which antibody react- small intestine and release , which enters intest- ing with the toxin modifies it so that it cannot bind to nerve inal epithelial cells and greatly increases chloride secretion EXPERT REVIEW OF VACCINES 5

leading to secretory diarrhea. Rehydration is an effective treat- (receptor blockade); antibody-mediated toxin neutralization ment and with proper care the mortality can be reduced to directed to LR and EF is also an alternative. less than 2% [20]. Safe drinking water is the key to prevention. Effective vaccination is directed to mucosal immunity. This is 2.2.4. Polysaccharide vaccines accomplished by oral administration of killed bacteria which The next group of organisms has in common a polysaccharide was introduced in the 1990s. A single dose oral vaccine is now capsule. Antibodies against the capsule can block infection. recommended for travelers and millions of doses have been Immunization with the purified polysaccharide induces a weak given [19]. Two oral vaccines are licensed. The Dukoral® mono- and temporary T-independent antibody response. A strong long- valent vaccine from Sweden contains formalin, heat killed lasting T-dependent response is induced using the polysaccharide whole cells of Vibrio Cholera O1 and a recombinant cholera antigen conjugated to a toxoid (diphtheria or tetanus). This toxin B subunit. The Sanchol/mORCVAX® vaccine from induces long-lasting IgG antibody which blocks bacteria adhesion Vietnam combines the O1 and O139 serogroups [19]. The … vaccines work by blocking the binding of the polysaccharide on the bacteria to epithelial receptors and inhibition of the protein , which is encoded within the genome of a 2.2.4.1. Haemophilus influenzae. Haemophilus influenzae filamentous bacteriophage, CTXϕ [20]. These vaccines provide (Hib) is a Gram-negative bacterium that primarily colonizes about 85% protection for 6 months and 50%-60% for the first the nasopharyngeal mucosa asymptomatically but can disse- year (WHO). An injectable vaccine is also available, but rarely minate to other organs causing , pneumonia, epi- used. glottitis and otitis media. Prior to use of vaccination Hib was a leading cause of childhood meningitis and pneumonia, causing up to 20,000 cases per year in the U.S. This has been reduced by 2.2.3. Anthrax 99%, eliminating Hib as a public health problem. The rate of Anthrax is a disease of hoofed animals (sheep, , etc.) invasive Hib disease decreased from 40 per 100,000 children to caused by . Humans are infected by contact fewer than 1 per 100,000 [26]. Hib is covered with polysacchar- with infected animals or their products. The manifestations of ide which allows it to be serotyped. Vaccines for Hib are direc- the disease are determined by the route of contact, cutaneous ted to produce antibodies to the capsule. However, after (skin ulcers), inhalation (acute pneumonia), gastrointestinal immunization with pure PSP antibody levels fall rapidly after (fever, vomiting, nausea) and (abscess) [21]. immunization [27]. The problem is that in this form the immune Death is common (20% in cutaneous and over 50% after response is T-cell independent. Since antigen processing by inhalation). The anthrax bacillus also exists in a spore form macrophages and T-cell help are required for a long-lasting where it is highly resilient to changes in environment or harsh response, new vaccines were developed by conjugation of chemical treatment. The characteristics of B. anthracis make it PSP to tetanus or diphtheria toxoid (Conjugate vaccines). This an ideal biological weapon [22]. Postal distribution of spores in principle was then extended to meningococcus and pneumo- 2001 led to antibiotic treatment of more than 30,000 poten- coccus, which have a similar pathogenesis. The immune tially exposed individuals. The pathogenesis of anthrax in mechanism is blocking of bacteria adhesion to mucous cells determined by the presence of plasmids that encode a tripar- by polysaccharides. However, Hib exists in two major forms: tite (AT) complex [23]: Lethal factor (LF, a metal- typeable Hib that which expresses serologically typable poly- loprotease), edema factor (EF, an adenylate cyclase) and saccharide (PSP) and non-typable HI (NTHi) which do not. NTHi protective antigen (PA-a ligand that transports LR and EF adheres to and colonizes the mucous membranes in the nasal into the cell). EF inhibits phagocytosis and causes edema; LF cavity through outer membrane proteins. Marked reduction in causes toxic shock and death. A live-attenuated form of B. Hib infections following wide-spread vaccination was followed anthracis introduced in 1881 by {23A}, was by an increase in H. influenza infections by NTHi, which do not refined, and is now used in China and Russia. This has been express PRP, presumably because of lack of competition for the discontinued in the western-world due to toxic effects such as infectious niche. To address this new threat, vaccines are being skin necrosis at the injection site [23]. Now refined culture developed to other outer membrane proteins (adhesions, por- supernatant from an attenuated strain that immunizes against ins, etc.) that perform a similar function for NTHi that PSP PA, known as AVA is used [24]. This stimulates production of accomplishes for Hib [28]. antibody to PA, and blocks toxin endocytosis. After three doses antibody appears at 6 months and with boosters leads 2.2.4.2. Meningococcus. Meningitis and sepsis are caused by to long-lasting immunity [23]. While the vaccine is effective, the bacterium Neisseria meningitidis; Phylum: Proteobacteria.It the time required to develop immunity is too long for defense is a round organism that tends to form pairs (diplococcus). against post exposure protection. It is recommended for There are approximately 3,000 cases of bacterial meningitis adults who are at risk for exposure: laboratory workers, those per year in the U.S. and about 330,000 world wide. About 10% exposed to infected animals, and some military personnel [25]. of adults carry the bacteria in the nasopharynx. The WHO New vaccines under trial are directed to capsule antigens such recommend vaccination for high-risk countries and for travelers γ as DPGA® and conjugates with PA, subunits of PA, - to these countries. Meningococcal vaccines have sharply derived PA and other approaches designed to enhance immu- reduced the incidence of the disease in developed countries nogenicity of PA, such as aluminum hydroxide absorbed PA. [29]. The bacteria colonize and penetrate the nasopharyngeal The vaccine blocks PA and inhibits toxin entering the cell mucosal membranes, pass into the bloodstream and are 6 S. SELL transported to the brain where they multiply in the subarach- recommended for adults 65 or over adults with health problems, noid space of the meninges. This is followed by increased smokers, and children older than two years with long term health permeability of the blood–brain barrier, trans-endothelial problems [33]. PCVs have 76 to 92% efficacy. The protective migration of inflammatory cells and release of cytokines and immune mechanism is receptor blockade and antibody-mediated prostaglandins [30]. Infected fluid from the meninges then opsonization (Immune complex). passes into the causing symptoms including stiff neck, fever and rashes. In some cases, there is bacterial sepsis 2.2.4.4. Streptococcus Group A (GAS). GAS are bacteria with vital organ failure; even with antibiotic treatment about commonly found in throat and skin and usually cause a mild 10% of patients will die. There are currently three vaccines illness, such as strep throat and impetigo, but can occasionally approved for use in the US. These are all quadrivalent targeting cause necrotizing fasciitis (flesh eating bacteria), toxic shock, serogroups A, C, W-135 and Y. One was licensed in 1981 and is acute rheumatic fever or rheumatic carditis [38]. composed of purified capsular polysaccharide for each of the Approximately 20% of patients with necrotizing fasciitis and four subgroups. The other two were introduced in 2005 60% with toxic shock die. About 10,000 cases of invasive Memacta® (Sanofi Pasteur) and 2010 Menveo® group A streptococcal occur in the US each year resulting in (GlaxoSmithKline/Novartis) and are conjugated to diphtheria about 2,000 deaths (NY.gov). Penicillin or erythromycin are toxoid. These are recommended for large scale effective treatments but do not cure each patient with inva- when an outbreak of meningococcal disease occurs. Several sive disease. Patients who recover from a GAS infection new vaccines conjugated to tetanus toxoid are also under develop antibodies to GAS M-protein and have protective investigation and one of these Men AWY-TT (Nimenrix®/Pfizer) immunity. Clinical trials with heat killed organisms and crude is recommend for travelers [31]. The vaccines are injected intra- M protein vaccines began in 1923 [39]. with little success. muscularly. Although still in use the polysaccharide vaccine is Following the development of rheumatic fever in 2 recipients no longer recommended and is being phased out. Conjugation receiving crude M proteins in 1969, further trials were prohib- to a carrier protein changes the immune response from T-cell ited by the FDA for 30 years [40]. Clinical trials with multiple independent (mostly low-affinity IgM antibody) to T-cell depen- peptides (up to 30) from different M serotypes induce bacter- dent to produce a long-lasting high-affinity IgG response [32]. icidal antibodies and are well tolerated and safe [40–42]. The objective is to produce complement-fixing antibody that Difficulties arise in assessing immunogenicity and tests such causes lysis or opsonization of the bacteria (immune complex as phagocytosis are difficult and tedious [40], More recently mechanism). common M-antigens have been genetically engineered to provide limited epitopes that will induce opsonic antibodies (immune complex) [43], but not autoantibodies [40]. Epitopes 2.2.4.3. Pneumococcus. Pneumococcal infection is caused by contained in the hypervariable, type-specific N-terminus of the a Gram-negative diplococcus: Streptococcus pneumonia.This M-proteins appear to meet these criteria [40,44]. Variations in organism lives naturally in the nasopharynx from where it can epitopes of M-protein have been extensively identified (emm spread to the lungs, brain and systemically as well as be trans- types) and may be used to design future vaccines for specific mitted to others [33]. The mortality rates of pneumonia are highly geographic areas. An alternative approach is to construct variable (from 1 to 45%) [34]. Invasive pneumococcal disease kills vaccines to the conserved C-region of M-protein and induce thousands of people in the US each year (most over 65) and is the IgA antibody (for example in the nose) that will inhibit binding most common cause of invasive bacterial infection in children. The to mucosa [45]. Additional vaccine candidates include pili, classic manifestation is lobar pneumonia with extensive consoli- fibronectin binding proteins [38], Protein F1/Sfb1, FbaA and dation of an entire lobe of the lung. Following the introduction of other bacterial products that bind tissues [40]. The immune vaccines, the overall invasive pneumococcal disease decreased mechanism of choice for therapy is antibody-induced from 100 cases per 100,000 in 1998 to 9 cases per 100,000 in opsonization. 2015 (CDC statistics). Of critical importance to the pathogenicity of S. pneumonia is its polysaccharide capsule which enables it to evade phagocytosis. Production of antibodies to this polysacchar- 2.3. Receptor blockade and T-CTL ide is the basis of the protection mediated by vaccination. The 2.3.1. Viral exanthems present vaccines target 13 or 23 serotypes of the bacteria Viral exanthems are skin eruptions produced by reaction of T- (Pneumovax® (PCV) 13 or 23) and new vaccines are under devel- cytotoxic cells with viral infected skin cells. Although starting opment that will increase the coverage up to 72 serotypes [34,35]. as a systemic infection, lesions appear in the skin correspond- The first vaccine was introduced in 1945 and included three ing to the appearance of T-CTL. Lesions can vary greatly in serotypes using purified capsular polysaccharides. As with other severity with heavily inflamed progressive necrotic lesions polysaccharide vaccines this vaccine is T-cell independent, pro- (small pox) to mild self-limited flat erythematous patches duces IgM antibody, short-term immunity and is ineffective in (Rubella). Small pox is named because the lesions are smaller children. To change the immune response to T-dependent the than the skin lesions of primary or secondary syphilis (large polysaccharides are conjugated to diphtheria toxoid which pox). Syphilis is not a viral exanthem. induces IgG antibody, memory B cells [36], and life-long immunity. Immunization with PCV 13 protects against fewer serotypes but 2.3.2. Smallpox has greater immunogenicity that PCV 23 leading to the suggestion Smallpox virus is in the family Poxvirus; genus: Orthopoxvirus; that both vaccines be used [37]. In the vaccination is species: Variola virus. It has a single linear double stranded EXPERT REVIEW OF VACCINES 7

DNA genome. Pox viruses are ‘proviruses’; they replicate in the inoculation and soon was widely practiced in Great Britain. cytoplasm of infected cells, not the nucleus. It has two forms During the revolutionary war the American army was deci- enveloped and unenveloped; both are infective. The viral mated by small pox, but the disease was controlled by variola- envelope is made up of modified Golgi membranes containing tion. However, because virulent virus was used, the disease viral specific polypeptides. This complicates vaccine design, became endemic and would never be eliminated [49]. because antibodies to the envelope that might block cell involves direct infection of the epidermis of the entry would not act on viruses without an envelope [46]. skin so that an immune response is initiated before systematic Before vaccination, the risk of death following contracting infection occurs [49]. Once inhaled, variola major virus invades the disease was about 30%, with higher rates among babies. the oropharyngeal (mouth and throat) or the respiratory Often those who survived had extensive scarring of their skin mucosa, migrates to regional lymph nodes, multiplies and and some were left blind. In eighteenth-century Europe, it is moves from cell to cell. By the 12th day or so, lysis of many estimated 400,000 people per year died from the disease, and infected cells occurs and the virus is present the bloodstream one-third of the cases resulted in blindness. Smallpox is esti- in large numbers (viremia). A second wave of multiplication mated to have killed up to 300 million people in the twentieth occurs in the spleen, bone marrow, and lymph nodes. The century and around 500 million people in the last 100 years of infection then spreads to the mouth and skin with formation its existence. As recently as 1967, 15 million cases occurred in of a skin rash that progresses to fluid filled bumps that rupture one year [47]. Vaccination against smallpox is one of the most and scar. By introducing the infection in the epidermis, the important advances in human preventive medicine. The terms immune response gets a head start on the systemic infection. vaccine and vaccination originate from the use of Cow-Pox as In 1796, , a simple country physician from an attenuated virus that induces Tcytotoxic cells that attack Gloucestershire, observed that Sarah Nelmes, a dairymaid virus infected epithelial cells. Vaccinia virus is closely related to developed a sore on her hand from Cow Pox infection became cow-pox virus and were once considered to be the same. The resistant to small pox [49]. Further observations on similar Latin name for Cow Pox is Variola vaccinae: the term vaccina- Cow Pox infections convinced Jenner that relatively benign tion was extended to include all forms of protective immuni- limited infection with Cow Pox was superior to variolation as zation. Vaccination against smalls pox is accomplished by fatalities were essentially eliminated and use of the attenuated introducing the attenuated virus into the epidermis with mul- virus removed the necessity to employ the virulent smallpox tiple needle punctures. If the vaccination is successful there is virus (variolation). Even though there was considerable resis- a crusting superficial contact dermatitis (e.g. Poison ivy) like tance to this [50], like the present reaction to measles and inflammation about a week after inoculation. This is termed a influenza vaccines today, The WHO launched universal vacci- ‘take’ reaction and indicated successful induction of immunity nation as the means to eliminate smallpox. The last know case to small pox. This process is so successful that small pox as a was reported on Somalia in 1977; the WHO certified the global disease has been essentially eliminated and vaccination dis- eradication of the disease in 1980 [47]. continued because of rare morbidity and mortality. However, stocks of the virulent virus remain in laboratories in the U.S. and Russia. The possibility of weaponizing smallpox has led to 2.3.3. Measles an increased interest in vaccine improvement [46]. In 2002, the Measles is a highly contagious systemic infection with the United States military resumed smallpox vaccinations as a main manifestation of multiple inflammatory lesions in the precaution to weaponized use. Since then at least 4 cases of skin (rash). Wild-type virus is spread though inhalation of life-threatening infections have occurred in individuals with respiratory aerosols. Infected macrophages spread the inflec- eczema who have had contact with vaccinated individuals tion to multiple organs (skin, kidney, lung and liver, etc.). The [47]. Historically smallpox probably was responsible for more skin lesions are a manifestation of T-cytotoxic cells invading deaths at early ages than any other disease. In epidemics up to and killing infected epidermal cells. (T-cell cytotoxicity). Before 50% of the children in a village might die. In ancient China vaccination measles was responsible for more than 2 million names were not given to infants until after they survived deaths annually; now about 100,000 globally. Although smallpox because so many children died that families would declared eradicated in the U. S. in 2002 [51], resistance of run out of cherished family names [48]. The Chinese noted some families to vaccination reduced herd immunity and by that milder forms of the disease resulted when individuals June 2019 over 1,000 cases were reported in the US. The were infected with virus from others with a relatively benign current vaccine (MeV) is an attenuation of wild-type virus by course of the disease. in vitro passage in human and chicken cells and is effective in About 1720, Lady Mary Wortley Monteque, the wife of the inducing both antibody and T-cytotoxic cells [52]. It is usually British ambassador to Turkey, observed this process and administered as a quadrivalent vaccine for measles, mumps, returned to England convinced of its efficacy [49]. She had rubella and varicella. The vaccine is effective in inducing both her 4-year-old daughter intentionally infected with virus from protective antibody to the H protein as well as CD8+ T-CTLs. a mild case of the disease and she was subsequently protected The antibody to H is required for blocking the reaction of the against small pox. She persuaded King George I to conduct WT virus with cell surface receptors (SLAM). Both antibody and trials on prisoners at Newgate Prison. This proved highly T-cytotoxic cells are necessary for effective immunity. The effective in preventing smallpox reducing mortality rates to vaccines are safe and effective and universal application as low as 1%. The process became known as variolation or could lead to eradication of the disease. 8 S. SELL

2.3.4. Rubella (German measles) limiting infection; but it could become life threatening in Rubella is a viral exanthem caused by the only member of the patients with T-cell immune deficiencies or rarely in normal togavirus family. The virus is encapsulated and has a single individuals. The first live-, vOka® was stranded RNA genome [53]. Most adult cases are mild with a developed by serial passage of the wild-type virus in generalized rash that occurs about 2 weeks after exposure, human and guinea pig embryo-fibroblast and human diploid lasts about 3 days and is less red than measles. It was first cells in Japan in 1974 [59]. In further trials it proved to be described in Germany and has become known as German safe and efficient [60] Current vaccines (Vairvax®.OKA/Merk) measles or three-day measles. Infection during early pregnancy and Varilrix® (OKA/GSK) are derived from vOka after passa- may lead to still birth or the congenital rubella syndrome (CRS, ging in cell culture and combined with measles-mumps- cataracts, deafness, heart and brain defects). The virus infects rubella-varicella live-attenuated vaccines [61]. This combined the placenta, spreads to the fetus and interferes with organ vaccine is >90% effective and has very rare side effects formation [54]. The nature of the receptor on infectible cells is [61,62]. The major protective immune response is IgA anti- not known, but it appears to be able to bind and infect various body inhibition of infection in the oropharynx; IgG antibody epithelial cell types. Rubella (from Latin ‘little red’) is spread can prevent spread of the infection by inhibiting binding to through the air though inhalation, rapidly disseminates and target cells and T-CTL by killing infected cells. can infect many other organs. In adults this is prevented by the immune response, but in fetuses there is dissemination to 2.3.6. Influenza many organs [53]. Only humans are infected; there are no Influenza is included in the category of viral exanthems because animal or bird carriers. The in a live-attenuated the virus infects bronchial epithelial cells and type II pneumocytes virus produced by passage in tissue culture. A single dose is which are then attached by T-cytotoxic cells leading to what could more than 95% protective [54]. Vaccination is highly recom- be called ‘viral exanthem of the lungs’ [63,64]. Influenza is caused mended for women of child bearing age. However, it is now by a highly mutable virus that is spread through inhalation of part of the standard childhood vaccination program and with contaminated air emitted from infected patients. It produces other viruses (MMRV). The incidence of rubella has dropped about 1 billion infections annually of which 35 million are severe, sharply following introduction of vaccination. During an epi- and up to 500,000 deaths. Vaccination is the primary approach to demic in the early 1960’s in the US rubella infections caused control and prevention. Because of transcription errors point 30,000 stillbirths and 20,000 children with CRS [55]. Since mutations result in different variations of surface antigens each vaccination was introduced the number of cases in the year with selection of the new variants as the susceptible popula- Americas dropped to 3,000 a year and the last reported case tion develops antibodies to the previous variants [65]. Vaccines are was reported in 2009. However, there are still about 100,000 either killed/inactivated or live-attenuated influenza viruses. Due cases of CRS per year in the rest of the world [54]. A vaccina- to emergence of different antigenic variants new influenza vac- tion rate of 80% should establish herd immunity and prevent cines are introduced each year to cover the new variants. The any further infections. Later after immunization or when the vaccines induce neutralizing antibodies to the globular head of exanthem appears adaptive active cell-mediated immunity is viral hemagglutinating and neuraminidase antigens. In the past, seen (increased TNF-α and IL-10) [56]. The exanthem indicates the vaccine contained 3 antigens (trivalent); quadrivalent vaccines T-cell cytotoxicity of infected epithelial cells, an effect that is are now being introduced to provide a wider range of protection. most likely taking place on other infected cells in the body. The live-attenuated vaccines are administered intranasally and Protective immunity is mediated by antibody neutralization of induce both local secretory IgA and circulating IgG antibodies, as receptors to block infection of cells and therapeutic immunity well as T-cell responses. However, the effectiveness of the live- by T-cell cytotoxicity of infected cells. attenuated vaccine has been questioned over the last few years and killed/inactivated viruses were recommended. A live-attenu- ated vaccine (FLUMIST®) was approved for the 2018–2019 flu 2.3.5. Varicella season [66]. Recently experimental models have defined the role Varicella zoster virus (VZV) is a member of the alpha herpes- of T-cell-mediated cytotoxicity asameansofkillinginfectedbron- virus family of DNA viruses. It has linear 125-kb double- chial mucosa and type-II pneumocytes analogous to the killing of stranded DNA genome encapsulated by a polyamine, lipid infected epidermal cells of the skin in viral exanthems [63,64]. An and glycoprotein envelope that binds to receptors on cells alternative to targeting surface molecules that are highly mutable and allows entry [57]. It causes two distinct diseases: chicken is to substitute internal structural proteins, which remain constant pox and herpes zoster (shingles). The virus initiates infection and induce both CD8 and CD4 T-cell responses. The induction of in the oropharynx and is transmitted systemically to skin cytotoxicity may be enhanced by using recombinant vaccinia virus epithelium via infected T cells [58]. Appearance of the typical as vector [64]. skin rash correlates with specific T-cytotoxic cells that target the surface glycoprotein and various viral proteins. These 2.3.7. Mumps cells are major mediators of epidermal injury and protect Mumps is cause by a paramyxovirus transmitted by inhalation. against future infection [58]. Decreased immunity with aging After a 12 to 15 days incubation period there is painful swelling allows recurrence of infection in sensory nerves (herpes of the parotid gland, the classic finding. (parotitis) that may zoster). VZV is ubiquitous and before a vaccine was available involve other organs such as meningitis, orchitis, oophoritis, essentially everyone developed chicken pox at some time in mastitis and pancreatitis. Historically mumps is a childhood infec- their life. In most instances this was a relatively mild self- tion [67,68]. Parotitis may not always be present and diagnosis EXPERT REVIEW OF VACCINES 9 depends on laboratory tests (RT-PCR for virus or IgM seroconver- Congo. It has proved to be 97% effective and almost certainly sion). Mumps epidemics occur about every 4–5 years mainly has prevented a global threat [74]. Even so there have been among children 5–7 years of age with about 6,000 cases per 1,206 cases and 764 deaths since 2018, but this is far less that epidemic in the US [69]. The disease is usually relative mild but the 2016 outbreak and logistical and safety problems. aseptic meningitis and orchitis can be serious complications. The Laboratory studies support the protective effect due to anti- disease is largely contained by vaccination. In the US the number body-mediated blocking of the binding of viral GP to host cell pf cases reported per year has declined from over 150,000 to surface receptors [75]. Since virus vectors are used for immu- about 300. The is a live-attenuated virus origin- nization, it is likely that there is also T-cell cytotoxicity. ally from the strains of mumps virus cultured in embryonic hens’ eggs and chick embryo cell cultures by 2.3.9. Rotavirus obtained from his daughter Jeryl Lynn when Rotavirus infects every child in the world and is the leading she had mumps in 1963 [70]. Two doses of the vaccine are 88% cause of severe diarrhea among young children, accounting effective in preventing disease, so in epidemics many patients for 20% to 30% of severe diarrheal disease that requires may have been vaccinated [71]. Other attenuated strains are hospitalization. There are more than 114 million rotavirus available, particularly in Russia, but are less effective and have diarrheal episodes, 24 million clinic visits, 2.4 million hospita- more side effects. The vaccine is believed to induce neutralizing lizations and 500,000 deaths per year in children younger than antibodies that block binding of the surface glycoprotein- 5 years of age [76]. Rotaviruses are large particles with three hemagglutinin-neuraminidase and fusion protein to glandular concentric protein layers that surround the viral genome [77]. target cells. T-cytotoxic lymphocytes to virus infected ductal cells The virus contains six structural proteins and six proteins that may also be necessary for complete protection [71]. are synthesized in the infected cell and function in viral repli- cation or interact with host protein to influence pathogenesis and the immune response. Viral proteins 7 (VP7) and VP4 bind 2.3.8. Ebola to small intestine enterocytes, contribute to serotypes and are Ebola virus disease is a viral hemorrhagic fever. There are four targets for blocking antibodies [77]. There are at least 11 VP4 viruses in the genus Ebolavirus that infect humans, named for types and 10 VP 7 serotypes, but 90% of infectious viruses the geographic areas first identified: Bundibugyo, Sudan, Tai belong to just four combinations. Rotavirus vaccines first Forest, and Zaire [72]. The Zaire virus, now commonly called became available in the US in 2006 and prevent 15–35% of just Ebola, is responsible for most human infections. The virus severe diarrhea in the developing world and up to 96% in the spreads through direct contact with contaminated fluids, such developed world. The vaccine should be used in high-risk as blood, saliva, vomit, sweat, etc. It replicates in many cells, areas and among food workers [78]. There are 6 rotavirus including endothelial cells, monocytes, liver cells, fibroblasts, vaccines that have been studied [69]. The most widely used etc. and causes apoptosis of immune cells and a weakened is RotaTeg®, which is a live, oral attenuated pentavalent vac- immune response. The virion begins infection by using a viral cine produced by recombination (FDA approved in 2006). An glycoprotein GP to attach to specific cell-surface receptors improved attenuated virus produced by multiple passages in such as Ctype lectin DC-SIAN followed by fusion of the viral culture (Rotarix®) produces long-lasting immunity after fewer envelope with cellular membranes. The virions are taken up by doses and is safe and effective in clinical trials: 85% effective the cell and then processed and the RNA transcribed into against severe diarrhea and 100% effective against the most positive strand RNAs. After infection, there is synthesis of severe cases [79]. Naturally acquired immunity correlates with Ebola virus glycoprotein which reduces specific integrins and intestinal IgA antibody and IgA memory cells. The vaccines are causes loss of vascular cell adhesion and hemorrhage. Left administered orally to facilitate the local IgA response. The untreated over half of those infected will die. One of the first major immune protective reaction is antibody neutralization outbreaks occurred in a village near the Ebola river in the of virus-target cell binding, but other mechanisms may con- Democratic Republic of the Congo. Since 1976 there have tribute including natural immunity and T-cell cytotoxicity. been regular outbreaks including the major one from December 2013 to March 2016 with 28,616 cases and 11, 2.3.10. HIV AIDS 310 deaths [72]. Organizations, including the WHO, NIH, The Human immunodeficiency virus (HIV) is a member of the ‘ ’ Gates Foundation and others funded a space race program to genus Lentivirus and family Retroviridae. HIV AIDS is trans- develop a vaccine for Ebola. In a relatively short period of time mitted by sexual contact or between a mother and fetus. It vaccines have been constructed and tested [73]. The first was continues to be an increasing world-wide plague. Although inactivated whole virus but this never went to trials because of retroviral therapy has resulted in a decrease of infections from safety and efficacy deficiencies. More sophisticated 3.3 million in 2002 to 2.3 million in 2012, there is a continued approaches utilize the envelope glycoprotein (GP) of EBV. increase in the number infected as therapy has resulted in The two leading candidates that entered clinical trials are many people living longer with subclinical HIV infections [80]. chimpanzee adenovirus 3 (ChAD3) and vesicular stomatitis Like Herpes, HIV infection is for life; it is not cured, only held in virus (VSV) vectors encoding the GP from the Ebola virus. check by retroviral therapy. Immunity to HIV is complicated by The VSV vaccine (rVSV-ZEBOV®) has proved to be both safe the fact that the target cells: CD4 T cells, macrophages and effective in ring vaccination clinical trials where both infected dendritic cells, are the same cells that take part in the immune individuals and others in the community are immunized. This response to HIV. Most effective vaccines are whole killed vaccine is currently being used to contain an outbreak in the organisms or attenuated organisms; killed HIV is not 10 S. SELL sufficiently antigenic and there are safety issues with a live individuals at risk, such as veterinarians or farm workers. One retrovirus vaccine. Two vaccine approaches are proposed: of the most important applications is the universal immuniza- antiENV neutralization and T-cell cytotoxicity [81,82]. The tion of dogs. This has been very effective in prevention of development of a neutralizing vaccine has proved to be very rabies by ‘herd immunity’. In the United States, rabies has complicated because of the genetic diversity receptor for been essentially eliminated by canine vaccination [85]. infectible cells and the nature of the HIV life cycle. Early trials Vaccines for rabies consisting of dried nerve tissue derived on inducing antibodies to selected HIV ENV proteins were from infected rabbits were introduced by Louis Pasteur and ineffective [83]. The next generation of clinical trials utilized Emile Roux in 1885 with initial success in treating a nine-year the AIDSVAX B/E envelope protein with a canary pox virus old boy bitten by a dog [86]. The virus produces 5 surface carrier (RV144). This yielded an efficiency of 31.2%; too low for proteins, the most critical for an immune response is the G licensing [83]. The diverse envelope (ENV) states involved in protein which functions for endocytosis and is highly anti- HIV-1 entry into HIV target cells pose a problem for vaccine genic [85]. The present vaccines are derived from inactivated, specificity. The proteins include a fusion envelope protein lyophilized virus cultured in human cells. The use of these closed state, a single CD4-bound intermediate, a three-CD4- vaccines has eliminated the necessity for multiple injections bound intermediate, a pre-hairpin intermediate and post- required by earlier vaccines which has markedly improved fusion states. These provide multiple epitopes for antibody patient participation [84]. All licensed vaccines have good specificity [82]. It is not yet clear which epitopes are critical safety records and are highly effective. The immune effector for receptor blocking antibodies and it is unlikely that a single mechanism is antibody-mediated neutralization (receptor antibody specificity would be effective. This understanding blockade) of the virus including local antibody production. came from study of hundreds of humoral antibodies that Production of rabies virus antibodies by B cells infiltrating were found in patients after five or more years of infection, across the blood–brain barrier is crucial for elimination of the termed broadly neutralizing antibodies (bNAbs). The confor- rabies virus from the brain [87]. In addition, T-TCL elimination mation of the ENV for optimal vaccine design for antibody of infected cells may be critical but may also contribute to loss neutralization is under investigation [83]. An additional of . approach is DNA in viral vectors (recombinant cytomegalo- virus, adenovirus and vaccinia virus) to induce CD8 + T-cytoxi- 2.3.12. Hepatitis city directed to infected cervical-vaginal mucosa [81]. It is 2.3.12.1. Hepatitis A. Hepatitis A virus is picornavirus that is argued that this local immunity could be critical to preventing passed by fecal-oral route in contaminated food and water. spread of HIV infection. Development of an effective vaccine Hepatitis A rarely causes chronic liver injury as does Hepatitis to HIV has a long way to go, but the resources and expertise B, but with few exceptions, produces an acute self-limiting being applied should be productive of an effective vaccine in illness. Like all hepatitis viruses, the virus has affinity for the the foreseeable future. It is likely that an effective vaccine will liver, i.e. it is hepatotrophic. Although during infection the need to include receptor blockade and CD8 + T-cell virus can be found in other organs, pathologic changes are cytotoxicity. limited to the liver and are the result of the host immune response to the infected liver cells [88]. There are both apop- 2.3.11. Rabies tosis and periportal piecemeal necrosis characteristic of auto- Rabies is an infection of the central by viruses immune hepatitis mediated by T-cytotoxic cells. Piecemeal of the Lyssavirus genus. Human infection usually follows a necrosis extends from the periportal region gradually across transdermal bite or scratch by an infected animal. the liver cord and consists of lymphocytes surrounding and Rabies in dogs is responsible of 99% of human infections killing individual hepatocytes. Plasma cells may also be seen in and poses a potential threat to 3.3 billion people. The inocu- areas of inflammation and antibody produced to the virus lated virus enters peripheral nerves and sometimes muscle results in long-term immunity after infection by receptor cells at the motor endplate of the neuromuscular junctions blockade. There is a very high incidence of hepatitis A in by endocytosis and is then transported to the central nervous endemic areas with most children asymptomatically infected system through motor neurons [84]. In the brain the virus before adulthood. Of these, about 1% develop liver failure. The replicates and then, after 1–3 months, it is distributed systemi- introduction of vaccines in the 1990’s has had a major bene- cally where it thrives in the salivary glands [85] and is available ficial effect. The estimated number of cases has declined by for transmission by biting. In the CNS, the infection results in 94–97% in the United States, but there still may be over 1.5 progressive fatal characterized by hyperac- million cases per year worldwide [89]. Vaccination works by tivity, fluctuating consciousness, and in severe cases, hydro- induction of circulating antibody that prevents infection of phobia and death by cardiac arrest. hepatocytes; again, these antibodies do not act on already Vaccination against rabies is applied both therapeutically infected cells. This requires T-cell cytotoxicity as is the case and preventatively. Because of the relative long latent period for other hepatitis virus infections. after infection, the vaccine is used to induce an immune response in infected individuals before the virus can propa- 2.3.12.2. Hepatitis B. This hepatotrophic virus has a high gate in the brain and cause symptoms. In some cases, if it incidence in endemic countries with poor sanitation (almost appears that it will take too long for the primary immune 120 million in the western Pacific region and 60 million in response to occur, immune globulin is recommended along Africa), with a much lower incidence in North and South with immunization. Vaccination is also recommended for America (less than 5 million) [90] During pregnancy it is EXPERT REVIEW OF VACCINES 11 frequently transmitted from infected mothers to their fetuses. mortality in pregnant women [98]. The immune response is In these children and those infected before 2 years of age, humoral neutralizing antibody directed against an open reading with an immature immune system, infection produces a frame (ORF-2) that encodes the antigenic capsid protein. A vac- chronic condition with liver failure and a high incidence of cine (Hecolin®) based on this antigen has proven 88% effective in liver cancer. Complex immune mechanisms lead to chronic China [98] but has not been commercialized because it is persistent infection [82], but a common feature is an attenu- thought that it will not be profitable [99]. The WHO has deter- ated T-cell response. In adults, an acute febrile illness, usually mined that further safety and efficacy studies should be done resolving but with rare liver failure is more common. HBV has before the vaccine is licensed [99]. both low- and high-affinity binding to hepatocytes which can be blocked by specific antibodies to HBV. The virus is not 2.3.13. Polio cytopathic; liver damage is mediated by T-CTLs reacting with Poliomyelitis is caused by an enterovirus that enters the naso- infected hepatocytes and features piecemeal necrosis. Four to pharynx and GI tract where it infects macrophages and over- 7 weeks after infection hepatitis B surface antigen (Australian lying epithelial cells in tonsils and Peyer’s patches that express antigen [91] may be detected in the serum whereas later in PV receptors (CD155) [100,101]. The virus replicates in these infection this is replaced by antibody to HBsAg. Circulating cells and are shed into the GI tract or carried to the central antibody has a role in suppressing chronic infection but does nervous system. From descriptions of the histopathology in not affect already infected cells. Patients treated with anti-B experimentally infected non-human primates [102], one can cell therapy (rituximab®) have a high risk of viral reactivation. speculate that T-cell-mediated activation of macrophages Vaccination has had a marked impact on HBV infection. (delayed hypersensitivity) eventually arms the infected macro- Because of the risk of neonatal infection, the WHO recom- phages and T-cell-mediated cytotoxicity is directed to the mends not only vaccination of mothers, but also newborns infected enterocytes to cure the gastrointestinal and systemic within 24 hours after birth. There are 2 effective and safe infection, but usually not before the virus has spread to the vaccines. The first was HBsAg isolated from serum of asympto- CNS. Circulating antibody induced by polio vaccination cannot matic carriers [92]. The second was from cloned HBV DNA act on the infected monocytes or epithelial cells but could fragments encoding the S protein [93]. It may be administered block infection of monocytes, and prevent the virus from separately or in combination with DPT and polio [89]. There spreading to the anterior horn cells of the spinal cord. There, are varied doses and schedules recommended but the vaccine TCTL to infected cells causes death and failure of these cells to is effective in infants, children and adults with high risk [94]. send signals to and flaccid paralysis. Prominent Even so, HBV remains a major public health problem world- is lymphocytic infiltration with eventual phagocytosis and wide and is the major contributor to liver cirrhosis and liver destruction of the infected cells [103]. In many other infectious cancer. In contrast, with application of an aggressive policy of diseases T-cell responses are key to clearing infected cells and universal vaccination for HBV there has been a dramatic recovery (for example, small pox, measles, influenza, syphilis, decline of acute HBV in the United States [89]. tuberculosis, etc.). In other infections, there is redundancy or the involved tissue cells have the capacity for rapid replace- 2.3.12.3. Hepatitis C. HCV is a major cause of morbidity and ment. However, there is no replacement capacity or redun- mortality worldwide accounting forover54,000deathsandalmost dancy in the large motor neurons of the CNS. 1 million disability adjusted life-years [89]. Therapy and prevention Paralytic polio is a disease of the twentieth century. Prior to are complicated by the fact that there are seven know genotypes widespread sanitation most newborn infants became infected [95] and there is rapid error prone replication in vivo resulting in with polio before appearance or myelination of the anterior production of numerous replicants that can avoid the host horn cells. In this instance antibody production occurred immune response and response to therapy [95]. There is no effec- before the anterior horn cells developed sufficiently to be tive vaccine and treatment depends on interferon and new direct infected. Before sanitation polio was relatively rare and did acting anti-viral drugs [89,95]. Both CD4+ and CD8 + T cells are not appear in epidemic form, usually occurring sporadically in required for vial clearance, and the role of antibody is not clear [96]. 2–4 years old children. On the other hand, in the twentieth There is a detectable antibody response within 1 month of infec- century, many children and young adults were not exposed to tion, but the intracellular virus is not affected by humoral antibody. the virus until after the anterior horn cell were sufficiently On the other hand, remission is associated with prominent CD4 mature to become infected. In the early twentieth-century + and CD8 + T-cell responses. Modified vaccinia and adenoviral widespread epidemics became frequent involving older chil- vectored vaccine encoding for proteins of HCV has been used to dren and young adults. By the twenty-first century, world-wide generate HCV specific T cells and clinical trials are now underway application of polio vaccines essentially eliminated the disease [97]. except for pockets in remote places usually afflicted by strife or religious objections to immunization [104]. Two vaccines 2.3.12.4. Hepatitis E. Hepatitis E virus has four genotypes, are available for polio, formalin killed (Salk) and attenuated with types 1 and 2 most likely to infect humans. The WHO live virus (Sabin). Both produce effective immunity, but each estimates that there are 29 million infections worldwide with has an advantage and disadvantage [104]. The attenuated 44,000 deaths, mostly in resource poor areas including China, virus in the Sabin vaccine infects gastrointestinal cells and North Africa and Central America. The virus is shed in the stool produces local immunity as well as systemic immunity. The resulting in fecal-oral spread through water contamination. The Salk vaccine does not induce local immunity so that even if disease usually resolves in 2–6weeksbutcancauseupto30% the individual is protected from neuronal loss, the wild-type 12 S. SELL virus can reproduce in the gastrointestinal tract, proliferate infected cells [108,109]. Chemotherapy for Chagas disease, benz- and spread to others; herd immunity is not established with nidiazole or nifurtimiz is highly effective if given early after the Salk vaccine. On the other hand, there is a low but infection, but becomes increasing ineffective as the infection significant morbidity and mortality with use of the Sabin progresses. There are two approaches to vaccines; preventive vaccine, but not with the Salk vaccine. and therapeutic [110]. Preventative vaccines could be directed to The effectiveness of the vaccines is unquestionable. The inci- blocking binding of trypomastigotes to cell surface receptors. dence of the disease in the United States dropped from over Therapeutic vaccines would need to be directed to CD8 + T-CTLs 22,000 cases per year to 4,594 after introduction of the Salk inducing a cytotoxic cell response to remove infected cells com- vaccine to 468 when both vaccines were used and from 1973 bined with chemotherapy [110]. Present pre-clinical vaccine can- to 1978 and with widespread use of the Sabin vaccine to nine didates include recombinant proteins, attenuated organisms, cases per year [105]. There are now approximately 400 cases per adenoviral and salmonella vectors. Since T-CTL responses year world wide, but also a few isolated incidences were reinfec- might be best for both preventative and therapeutic vaccines, tion is imported [104]. Because of rare infections events following cutaneous administration of a vaccinia-vectored vaccine should oral poliovirus vaccine (OPV), most countries have shifted to be considered. inactivated polio virus vaccine (IPV). With the overwhelming success of polio vaccination world wide more cases are caused 2.3.15. Epstein Barr virus by OPV than by wild-type virus. The WHO, the United Nations Epstein-Barr virus is the causative agent of infectious mononucleo- ’ Children s Fund and Rotary International developed a Polio sisaswellasbeinglinkedtomalignancies, including nasophar- Eradication and Endgame Strategic Plan to wipe out polio by yngeal carcinoma, gastric carcinoma, Hodgkin disease, Burkett the year 2018 (the original goal was by 2000). According to the lymphoma and lymphomas in immunocompromised persons. WHO as of March 2019, 33 cases were identified in 2018. The virus infects B cells in the tonsils directly or after replication Therefore, it is so close, yet so far away. It is likely that the IPV in epithelial cells in the oropharynx. The major clinical effect of will continue to be given for some time until there are no more infectious mononucleosis is self-limited debilitating fatigue. It cases of WT infection. At this time, there is continued fine tuning affects about 500 out of 100,000 persons each year in the United of vaccine type, dose and schedule to determine the best way to States [111], with about 20% with persistent manifestations caus- maintain complete eradication [104]. ing loss of work time. Most of the malignant manifestations are due to proliferation of B cells (B-cell lymphoma). The diagnostic 2.3.14. Chagas disease large mononuclear cells (mononucleosis cells) seen in the blood Chagas disease (American trypanosomiasis) is an inflammatory are not malignant but monocytes reactive to the B cells. This cardiopathy caused by Trypanosoma cruzi transmitted by kissing implies a DTH response with CD4 + T-cell-mediated activation of bugs (reduviid bug). In addition to humans, natural hosts include macrophages. Vaccine candidates include EBV glycoproteins, lytic domestic and wild animals. In Latin America, there are an esti- proteins, and latent proteins. The primary candidate has been EBV – mated 9 10 million infections per year with over 10,000 deaths glycoprotein gp350 through which EBV binds to CD-21 on B cells and 100,000 years lived with disability [106]. About 70% of [112]; antibody to gp350, which acts to inhibit infection (neutrali- infections are asymptomatic, in the remaining there may be an zation mechanism) or as a target for cytolytic T cells. T-cytotoxicity – asymptomatic stage lasting 4 8 weeks, but in some there is a may limit the first stage of infection of the epithelial level and may localized swelling at the infection site (Chagoma). Disease man- block reactivation of proliferation in B cells [113]. This is especially ifestations include arrhythmia, aneurysms and heart failure and/ important in immunosuppressed transplant patients where infec- or gastrointestinal dilation and hypertrophy. Death is usually due tion arises from the EBV-infected transplanted organ. Potential T- to ventricular tachycardia or fibrillation [107]. Trypanosoma cruzi cell immunogens include EBV early response proteins Zta and Rta enters the host through deposit of contaminated feces into fresh [114], lytic proteins, structural proteins [115], or EBV latent proteins bite sites or mucosal surfaces as trypomastigote forms, and expressed on infected B cells, such as EBV nuclear antigen (EBNA1) infects epithelial cells, fibroblasts and macrophages. After initial and leader protein (EBNA-LP). A Phase-II trial of gp350 in alum/ rounds of replication near the site of inoculation the released MBL reduced the rate of infectious mononucleosis by 78% [115]. trypomastigotes infect a wide range of cell types throughout the Two new vaccines have been developed; one to elicit antibodies body [107]. The parasites develop into the epimastigote form in to gH/gL, a protein on epithelial cells that fuses with EBV [116], and the midgut. These multiply and are released back into the colon another against a viral protein (gp42) that enhances fusion of the as trypomastigotes where they are passed into the feces. During virus with B cells [117]. These approaches have not yet been tested infection the parasites show affinity to the heart and can multiply in clinical trials. massively there. This causes damage to myocardial fibers and intense cellular inflammatory infiltrate changing from acute to chronic featuring T cells and macrophages and non-necrotizing 2.4. Receptor blockade and DTH granulomata with giant cells. Affected hearts become markedly enlarged with aneurysm formation. The progression of cardiac These are grouped together because there is a common pro- lesions continues even as the number of infecting organisms tective strategy: preventive, antibody-mediated receptor decreases, but some patients develop cardiac autoimmunity blockade (neutralization) and therapeutic (CD4 + T-cell- [107]. In addition to natural immune mechanisms, Trypanosoma mediated delayed hypersensitivity, DTH). However, differences cruzi infections feature Th1-type CD4+ helper cells with promi- in the pathogenesis and immune response reflect variations in nent development of CD8 + T-cytotoxicity responses that clear the natural history of the infection EXPERT REVIEW OF VACCINES 13

2.4.1. Yellow fever by endocytosis and passed to neurons [122]. Humans are dead- Yellow fever is caused by a member of the RNA Flavirviridae end hosts for the virus; infected humans cannot transmit the family which includes yellow fever, Japanese encephalitis, virus to another mosquito. The natural animal hosts are birds and West Nile and dengue viruses. These are transmitted by mos- mammals, especially pigs. Although very common in endemic quitoes in endemic areas. Upon inoculation into the skin the areas only about 1% of infected individuals develop clinical virus disseminates to the draining lymph nodes where follow- disease [123]. There are 30,000 to 50,000 cases per year with a ing clathedrin-dependent endocytosis into mononuclear cells, death rate of 30% [124]. Culex mosquitoes live throughout particularly monocytes, the genome is rapidly translated, pro- South-East Asia and in the tropics, Middle East and Africa. cessed and productive of infective virus [118]. Then the virus Because there is no effective anti-viral treatment and prevention reaches the liver and infects hepatocytes, most likely mediated by elimination of the carrier mosquitoes is difficult, vaccination is by infected Kupffer cells, and causes apoptosis of the liver, considered as the most effective way of preventing the disease. accentuated by NK cells [119]. After a short incubation, symp- Various vaccines, including inactivated mouse brain derived toms include fever, muscle pain and , which usually virus, live-attenuated viruses, and formalin-inactivated virus, improve within 5 days. However, after this resolution symptom which had acceptable, but not safe or effective have been tested. recurs more severely in about 15% of infected individuals and The present IC51 vaccine is a purified, inactivated whole virus liver damage leads to yellow skin (jaundice) and about half of (IXIARO®). It was derived from an attenuated virus (SAL14-14-2), those with recurring illness will die [120]. Historically, yellow cultivated in Vero cells and formulated with 0.1% aluminum fever became prominent in the nineteenth century [121]. It hydroxide. This has proven to be safe and 90% effective [124]. was introduced into South America through the slave trade in At present, the vaccine is underutilized and there are recommen- the 1800’s and the higher incidence in the disease in slaves dations to expand use in endemic areas and travelers to endemic when compared to Europeans led Napoleon to sell the areas [125]. The vaccine induces high titers of neutralizing anti- Louisiana Territory to the United States in 1803, with a massive body that prevents infection of susceptible cultured cells. A key epidemic occurring in Memphis, Tennessee in 1878. Soon after antigen is NS1, a glycoprotein secreted during viral replication. this Walter Reed demonstrated that the disease was carried by Anti-sera to NS1 prevents experimental infection in mice [126], mosquitos. Control of the disease by elimination of mosquito but CD8 + T-cell cytotoxicity and CD4+ activation of macro- transmission greatly facilitated the construction of the Panama phages (INF-γ) may also be active in recovery from infection Canal [121]. The first vaccine to yellow fever was developed in [127]. In any case, the major effect of vaccination is to prevent 1927 from attenuation of wild-type virus by passage in mouse infection by blocking virus binding to host cells. brain B cells. In the 1940s, such a strain, called the French strain, was developed at the Institute Pasteur and used exten- sively in French West Africa, but it’s association with post- 2.4.3. Dengue fever vaccinal encephalitis led to discontinuation in 1981. In the Dengue fever includes a high fever, , vomiting, mus- meantime, another strain, the Asibi strain, named after the cle and joint pains, and a characteristic skin rash. It begins patient from which it was isolated in Ghana, was used at the about 2 weeks after being bitten by an infected mosquito Rockefeller Institute to produce an attenuated stain after [128]. About 80% of exposed individuals have no symptoms. mouse and chick cell culture and called 17D in 1945. This Upon injection the virus binds to and infects Langerhans cells vaccine has been given at least 600 million times since then in the skin which migrate to draining lymph nodes. The infec- and a single dose at about 9 months of age yields 80–100% tion is passed on to circulating monocytes where the virus efficacy [119]. Wide spread vaccination campaigns in Africa multiplies [128]. Many of the symptoms of Dengue fever are have resulted in the almost complete disappearance of YF believed to be due to cytokines and interferons released from and currently 35 or the 44 countries at high risk have routine these infected cells. Unusually severe forms include dengue childhood immunization programs, making these areas more hemorrhagic fever and dengue shock syndrome. The Dengue habitable [118]. The major mechanism of vaccine-induced virus has 4 serotypes for which a vaccine must be directed. immunity is antibody-mediated viral neutralization, but cellu- Several vaccines are under investigation, including live atte- lar mechanisms, such as CD4+ DTH induced by immunization nuated, inactivated, DNA and subunit vaccines. Live-attenu- to 17D, may stimulate CD4 + T cells to activate infected ated vaccines are the furthest along [129]. In 2016 Dengvaxia®, macrophages [118]. A rare but often severe and fatal reaction a live-attenuated tetravalent vaccine made by recombinant to 17D vaccination is yellow fever-associated viscerotrophic DNA processing was used in high-risk countries with partial reaction (YE-AVD) and efforts are underway to identify those success. A major problem is that seronegative recipients have at high risk for this reaction to improve safety [121]. a high risk of severe dengue after immunization, so it is only recommended for seropositive individuals, essentially acting as a booster immunization for those who have recovered from 2.4.2. Japanese encephalitis Dengue. Ongoing trials in Latin America and Asia reveal an Japanese encephalitis is caused by a single-stranded RNA virus efficacy of about 60% in seronegative recipients and over 80% that is transmitted by the Culex mosquito. The virus enters the in seropositive. Several other vaccines are under development via infected lymphocytes or monocytes [130]. Since the virus replicates in monocytes and not epithe- where the virions bind to the endothelial surface, are internalized lial cells immunization that favors DTH (BCG vector) might be 14 S. SELL effective in activating the infected monocytes to eliminate the 2.4.6. Zika virus infective viruses. Zika virus is a mosquito-transmitted virus in the Flaviviridae family of RNA viruses. It was first identified in a monkey in the Zika forest of Uganda. Zika virus emerged as a clinically significant problem in 2.4.4. Chikungunya virus 2015 when the virus spread to South America causing congenital Chikungunya virus (CHIKV) is a positive sense RNA togavirus trans- malformations and microcephaly and Guillain–Barresyndromein mitted by Aedes mosquitoes. It causes an acute febrile illness adults [141]. By 2018 over 800,000 cases were reported in the followed by debilitating polyarthralgia of the peripheral joints Americas, including the U.S. Five vaccine designs are currently like Dengue virus [131]. Prior to 2004, CHIKV was an endemic under investigation: DNA vaccine, purified inactivated virus, live- disease in Africa and Asia that produced periodic outbreaks. attenuated virus mRNA vaccine and viral vector based [142]. There Since then large urban epidemics have occurred in India and the are multiple epitopes of the envelope proteins (E) that could be virus has been detected in the Western hemisphere. In 2014, there targets for neutralizing antibodies [143]. The vaccines include the were infections in 43 countries in the Americas involving over 1.1 E and PrM proteins which make up the outer protein coat of the million people and it is estimated that there are over 3.4 million virus virion [141]. Experimental studies demonstrate that the vac- people infected globally [131]. There is no approved vaccine. In cinesinduceblockingantibodiesaswellasCD8+T-CTLtostruc- non-human primate models, infection stimulates neutralizing anti- tural proteins [143]; both are desirable for protective immunity. bodies as well as reactive T cells [132] and administration of a There is no approved vaccine and many problems of evaluation of human recombinant monoclonal antibody to infected Rhesus effectiveness and possible adverse outcomes remain. macaques caused rapid elimination of the virus and less severe joint disease that in control treated animals [133]. Humoral anti- bodies primarily target the structural glycoproteins on the virions 2.4.7. Typhoid fever surface (E1 and E2) and these are the targets for most vaccine Typhoid fever is caused by Gram-negative bacteria in the strategies [131]. The first vaccine tested was a formalin-inactivated Salmonella family (Typhi and parathyphi). These are found only in derived from viruses cultured obtain from infected suckling mouse man and are transmitted by ingestion of contaminated food or brains and later from in vitro cultures at Walter Reed Army Institute. water. The organism penetrates the small bowel epithelium and More recently inactivated virus obtained directly from an infected spreads by lymphatics to the blood stream. It may be treated with patient and live-attenuated virus have been tested in human antibiotics but increasing incidence of drug resistant strains has clinical trials and effectively induce neutralizing antibodies [131]. made prevention by vaccination more important than previously. Unfortunately, these approaches were discontinued in 1998 Untreated about 20% of those infected will die [144]. Sanitation due to limited funding and the unpredictable outbreaks of the during food preparation is the most effect way to prevent the diseases [134]. A positive response correlated with INF-γ,apro- disease. While the disease is rare in developed countries, it is duct of activated CD4 + T cells (DTH), in an experimental mouse particularly high in Asia and part of Africa. In the US, the vaccine model [135]. Active investigation of a variety of vaccine options is is only recommended for those at high risk, such as travelers to now underway including virus like particles, subunit vaccines, endemic areas [145]. There are multiple immune effector mechan- vectored and chimeric vaccines, nucleic acid vaccines and live- isms in response to infection with S. typhi or S. paratyphi infection: attenuated vaccines [132]. Clinical trials are limited by lack of first, natural immunity in the form of IFN-γ producing natural killer funding. Because DTH most likely is critical for a therapeutic cells and then adaptive immunity. There are antibodies in both vaccine, a recombinant BCG vaccine should be considered. blood and intestinal secretions to both cell surface and flagellar antigens (O, Vi, and H) with evidence of both systemic and local 2.4.5. West Nile fever (Peyer’s patch) antibody production, as well as activation of West Nile virus is transmitted by mosquitos from infected birds to CD4 + T cells. Since the organisms infect cells in the monocytic animals, including dogs, horses, cats and humans [136,137]. About series both in the intestine (M-cells) and lymphoid tissue (mono- 20% of humans exposed develop fever, fatigue and weakness only, cytes) it is likely that T-cell-mediated activation of macrophages but a small number develop a severe infection that can last for (delayed hypersensitivity) would be active. The first typhoid vac- months and produce severe brain damage [138]. There is no cines were developed in 1896. Since then there have been many approved vaccine; the best method to prevent the spread of the improvements [145]. There are several safe and effective widely disease is to avoid mosquitos. Also, since birds are the primary used vaccines: Vi capsular vaccine (Typhim Vi®®) injected intra- reservoir for the virus, risk can be evaluated by the presence of the muscularly; Ty21 oral vaccine (Vivotif®) and combined with hepa- virusindeadbirds.AswithotherFlaviviridae, after being bitten by titis A and Vi polysaccharide (ViVaxin® and Hepatiyrix®). The cap- an infected mosquito, the virus enters and replicates in dendritic sular polysaccharide vaccines are isolated from the Ty2 strain of S. cells and monocytes. These infected dendritic cells may inhibit NK typhi and have limited T-cell responses and booster effects [146], cell protection and other T-cell functions [139]. There is no but are effective in producing IgM antibodies. The oral vaccines do approved vaccine for humans, but a vaccine called not contain VI antigens but promote a local mucosal immune ChimerivaxWNV® is under development and in stage II clinical trials response in the GI tract with IgA antibody as well as systemic [140]. Again, in addition to receptor blockade to prevent infection, IgG antibody and T-cell activation [147]. Conjugate and live-atte- consideration should be given to induction of DTH to activate nuated vaccines are under development [146]andhavegreater infected macrophages to kill intracellular organisms. potential for T-cell activation. EXPERT REVIEW OF VACCINES 15

2.5. Cytolytic 2.5.1.2. Whole organisms. This approach is based on the finding that sporozoites killed by irradiation could result in 2.5.1. Malaria sterile protection after homologous infection in about half of The malaria parasite, Plasmodium falciparum, caused 219 mil- immunized adults (for summary of result [148]. A more pro- lion infections and 435,000 deaths in 2017, essentially the mising approach now in field trials is to combine an attenu- same as in 2016 (212 million infections and 429 deaths [138]. ated infectious parasite with deletion of three genes that These figures have been even higher. Between 2000 and 2013, arrests at a late stage in the liver with an antimalarial drug. the incidence fell by 30% and mortality by 47% due to pre- ventive actions such as distribution of bed nets, Indoor resi- dual spraying and artemisinin-based therapies [138]. Future 2.5.1.3. Blood-stage vaccines. These are directed to redu- gains against malaria depend on availability of an effective cing clinical illness. These are directed to merozoite surface vaccine [139]. This is complicated by the complex life cycle of antigens and a red cell membrane protein which contributes P. faliciparum as well as other malaria causing strains. The to RBC invasion. When combined with a potent liposomal infective stage of P. falciparum, (sporozoites) is carried by the adjuvant this was more that 60% effective in one test in female Anopheles mosquito. Upon biting a human, the spor- children, but not in another test in adults [148]. Tests are ozoites infect hepatocytes where they multiply and develop ongoing with a variety of similar approaches including one into merozoites. These are released into the circulation and for P. vivax. The immune mechanism is antibody-mediated cell infect red blood cells, where they produce symptoms by lysis (cytotoxic). destruction of RBCs. A small number mature to male and female gametocytes which, when taken up the female mos- 2.6. Atopic quitos during a blood meal, will reproduce to form sporo- zoites, completing the life cycle [148,149]. Vaccines may be 2.6.1. Helminths directed to the pre-erythrocyte stage, blood stage, or sexual Helminthiasis is an infection by worms including tapeworms, stage. Erythrocytes lack major histocompatibility antigens and flukes and roundworms. These worms usually live in the GI serve as a protective niche for merozoites against T-cell- tract but may burrow into other tissues where they induce mediated mechanisms but do express parasite antigens that inflammation. The eggs or the worms are found in soil and are can be recognized by antibody. Thus, antibody-mediated cyto- taken in orally. The infection leads to poor development, toxicity is the major mechanism for immune defense against malnutrition and anemia. During helminth infections, there malaria. The antigens expressed on the infected RBCs reflect are elevated serum levels of IgE, tissue eosinophilia, mastocy- genetic variations of the organisms to avoid this immune tosis, and Th2 helper cells. These cells produce IL-4, IL-5, and attack. An effective vaccine must elicit antibodies to a variety IL-14 and are believed to mediate protection against helminth of possible plasmodium antigens. Vaccines for malaria have infection [151]. Schistosome eggs in the liver or intestine been under development for over 50 years [148]. These are induce a strong eosinophil-rich inflammatory granuloma. directed to the pre-erythrocyte antigens, whole organisms and Other atopic reactions associated with infection are systemic blood stages. anaphylactic reactions, eczema and asthma [152], especially if infected early in life [153]. The WHO estimates that over 2 billion people are infected with soil-transmitted helminthiases, 2.5.1.1. Pre-erythrocyte vaccines. Vaccines for the liver including schistosomiasis, trematodiasis, filariasis, onchocercia- stage of infection would prevent malaria infection so that sis and echinococcosis [154]. These infections appear to be merozoites are not produced to then infect erythrocytes. This able to suppress an immune response through T-cell regula- vaccine is directed to the circumsporite surface protein (CAP). tory mechanisms [135]. At the present there are no effective The vaccine consists of a central repeat region (R0), T-cell vaccines for human helminth infections. In animal models, epitopes (T) and a carrier matrix of HBV surface antigen vaccination is successful in inducing worm expulsion [155]. (HBsAg) as a fusion protein co-expressed in Accharomyces Radiation attenuated larval vaccines are effective for bovine cerevisiae along with free HBsAg [150]. This is combined with lung worm and dog hook worm. There are three problems: an adjuvant from an oil-and water- emulsion of monopho- identifying the protective antigens, the immune effector sphoryl and a saponin derived from the South mechanism and an immunization protocol [155,156]. There is America soap bark tree Quillaia Saponaria. If effective the consensus that the immune effector mechanism is atopic immune response would induce T-cell cytotoxicity to kill (Th2), but it is not clear how to direct immunogen to this infected hepatocytes. In field tests in Africa this proved up to pathway. This may be done by selecting the appropriate 50% effective with limited side effects [148]. The vaccine is adjuvant; for example: diethylaminoethyl (DEAE) dextran about 30% effective in preventing infection in infants and in [157]. Human trials are now underway. April of 2019 has been approved for this use. The rationale is that even though the effectiveness is low, because of the large 2.6.2. Schistosomiasis number of infections in newborns and infants, even this effec- According to the WHO, Schistosomiasis is the third most devas- tiveness may be significant in lowering infection rates. tating tropical disease globally, and a major cause of morbidity However, questions regarding the influence of this vaccine and mortality. Although all 5 species can be treated effectively on shift of infection to other strains remain. Many other pre- with high doses of praziquantel [158], reinfection is frequent, erythrocyte antigens and adjuvants are under study [138]. and chemotherapy has limited value for prevention. Clearly, a 16 S. SELL vaccine is needed for effective control. However, development in blocking receptors effective for other infections, but these of a vaccine for schistosomiasis has proved to be elusive and have not proven to be effective in humans [167]. Two vaccines faces complications because of the complex life cycle of the have been tested: StaphVAX® (polysaccharide-protein conju- worm, which includes several distinct phases. Humans are gated) and V710® (to a cell wall protein). The first proved to be infected through the skin through fresh water contaminated ineffective and the second increased infections in a surgical with free living cercariae derived from infected snails population. It is the conclusion of workers in the field that [159,160]. Within 2 days the cercariae changes form and bur- single antigen vaccines will not work so that a combined rows through the dermis, enters draining blood vessels, vaccine to increase both opsonizing and toxin neutralizing migrates to the lungs and then to liver where they feed on antibodies should be developed and tested [166]. One now red blood cells, mature and mate. Male-female worm pairs under development is SA4Ag®. This contains 4 antigens: the form and settle in either the portal or pelvic vessels, copulate adhesion molecule ClfA, the manganese transporter MntC and in situ and lay eggs which are released into the stool or urine antiphagocytic capsular polysaccharides 5 and 9 [168]. This is [161]. Because of the various forms that emerge during this life designed to elicit broad antibody and cell-mediated immunity cycle up to 20 different potential immunizing antigens have against multiple virulence factors and results of a Phase-I trial been identified; three of which: SM14, Sh28GST, and AM-TSP-2 show a robust response to all components of the vaccine have entered clinical trials [162]. Since there are no specific [166]. Other vaccines against surface polysaccharides, proteins, receptor-ligand interactions and no intracellular infections, and secreted toxins, as well as passive monoclonal antibodies potentially neutralizing (receptor blocking) antibodies would are in various stages of development [166]. not be expected to contribute to defense. In tissues eggs elicit a cellular reaction consisting of eosinophils, lymphocytes and 2.7.2. Lyme disease granuloma formation with hyperplasia of overlying epithelium Lyme borreliosis is a serious multisystem disease affecting the [159]. The inflammatory response includes Th2-type cytokines skin, joints, nervous system and heart caused by a bacterium [163] suggesting an atopic hypersensitivity reaction with gran- named Borrelia [169]. It is spread by infected ticks of the uloma formation consistent with poorly degradable antigen. genus Ixodes, which is common in the North Eastern U.S. It Production of antibody may result in a serum sickness-like is estimated to affect 300,000 people a year in the United illness known as Katayama syndrome [164]. In chronic disease States and 65,000 in Europe [170]. Lyme disease progresses in Th2-type response (IL-4, IL5, and IL-13) contributes to fibrosis three stage [171,172]: Stage-I cutaneous rash (erythema and morbidity [163]. It is not clear that the atopic mechanism migrans) with lympho-plasmocytic infiltrates around dermal is effective in combatting the infection. The problem is that vessels. Stage-II lympho-plasmacellular infiltrate in seen in the the atopic response may be desired for expulsion of worms meninges, ganglia, peripheral nerves and in heart in patients from the intestine, but this also contributes to chronic tissue with cardiac lesions. Stage-III hypertrophic synovitis and cuta- damage to eggs in tissues. An alternative mechanism might neous lymphoid hyperplasia are also seen. Erythema migrans be DTH which could effectively phagocytose eggs and larvae. begins as a red macule expanding to an annular lesion from 5 As suggested for syphilis vaccine (see below), a recombinant to more than 60 cm within days or weeks after infection [173] BCG vaccine may be the best approach to induce the desired often with fever, fatigue and headache. About two-thirds of delayed hypersensitivity response for a schistosomiasis untreated children develop arthritis without skin lesions and vaccine. 20% neurologic manifestations (nerve palsy) with increased lymphocytes in CSF [173]. Diagnosis may be confirmed by 2.7. Immune complex seroconversion. Organisms in in the dermis activate proteases and outer surface proteins selectively interact with endothe- 2.7.1. Staphylococcus aureus lial cells, platelets and extracellular matrix by interactions with Staphylococcus aureus is a Gram-negative bacterium that is an integrins, glycosaminoglycans, fibronectin and collagen pro- asymptomatic resident of the nasopharynx and skin. Upon moting heart and joint homing and colonization [172]. The invasion beyond the epidermal protective barriers, S. aureus role of the immune response appears to be protective as SCID causes skin and soft-tissue inflammation as well as septic mice which lack B and T cells develop severe arthritis and arthritis, pneumonia, septicemia, toxic shock, etc. carditis [171]. Initial spread of infection may be partially lim- Staphylococcus aureus infections were once well controlled ited by the natural immunity. B burgdorferi release molecules by treatment with methicillin, but drug resistant strains rapidly that produce natural immunity responses which may limit the appeared and now methicillin-resistant S. aureus (MRSA) is the infection and enhance the adaptive immune response, but leading cause of nosocomial infections world wide [165]. In also contribute to tissue damage. The specific immune the U.S., there are 300,000 hospitalization for S. aureus per year response may involve different immune mechanisms as early which accounts for up to $14.5 billion in hospital costs. arthritis is characterized by neutrophil infiltration, whereas Staphylococcus aureus produces acute necrotic infection later arthritis and carditis features macrophages and T-lym- (abscess). The adaptive immune defense is antibody-mediated phocytes suggesting a DTH reaction. The organisms are extra- opsonization (immune complex reaction). Essentially all adults cellular so that receptor blockade by antibody and T-CTLs are have antibodies to S. aureus surface antigens, but these do not most likely not involved in protection, but antibody to outer always fix complement and do not provide protection against membranes may neutralize the defense mechanism of the infection [166]. Many vaccine candidates have targeted cell organisms. The organism produces a variety of biologically surface polysaccharides or cell wall proteins that are effective active molecules that could be targets for inactivation and EXPERT REVIEW OF VACCINES 17 outer membrane proteins, but the most promising for a vac- 10 million people developed TB with 1.3 million deaths in HIV cine are outer surface proteins OspA and OspC, which are negative individuals and an additional 300,000 in those with involved in protecting the organism as it travels from the HIV [183]. The infection rate and deaths are much higher in infected tick to the host [174]. A vaccine based against developing countries than in high-income countries with OspA (LYMDrix®) was introduced and tested in clinical trials increasing cases caused by drug resistant strains. to have an efficacy of 76% in adults and 100% in children. About 23% of the world’s population has latent TB. The However, many recipients developed autoimmune and other protective response to infection with Mycobacterium tubercu- side effects, and although the CDC found no connection losis is macrophage activation by immune reactive CD4+ Tcells between the vaccine and the complaints, subsequent law (DTH). Mycobacterium tuberculosis infects macrophages. TB suits forced the vaccine to be withdrawn. In April 2019, con- specific CD4 + T cells can recognize the TB antigens on gress provided NIH with $6 million for prevention proposals macrophages and release cytokines that activate macrophages for Lyme disease [175]. The best prevention is avoidance of to kill the organisms. Unfortunately, this approach may not be ticks and removal from the skin of any ticks as soon as found. effective or there may be large numbers of organisms that Antibiotics given early after infection can prevent develop- cannot be removed by the macrophages. If the organisms and ment of symptoms. infected cells cannot be eliminated during the process of delayed hypersensitivity, surrounding macrophages are 2.8. T-CTL attracted and form a barrier around the infected tissue known (granuloma). Although the granuloma ‘walls off’ the 2.8.1. Human papilloma virus (HPV) infected cells; with age the granuloma may break down allow- HPV is one of most common sexually transmitted infections. ing residual viable organisms to disseminate from degenerat- Most HPV infections are symptom-free and resolve sponta- ing macrophages. HIV infection plays a major role is loss of neously [176]. There are over 100 HPV types, low-risk HPV this protection to tuberculosis and the reactivation rate in very types produce genital warts, whereas high-risk types cause high in patients with HIV. cervical cancer [177]. It is estimated that 5% of all cancers For vaccine-induced immune protection, humoral antibody world-wide are related to HPV infections [178], including cer- and T-cell cytotoxicity are ineffective and vaccination should vical, penile, anal, and oropharyngeal cancers and that up to be directed to producing DTH. For DTH CD4 + T cells release 20% of men carry one or more of the pathogenic HPV types cytokines that activate macrophages to kill the organisms in [179]. In 2006 the FDA licensed the first HPV vaccine directed their cytoplasm as well as arm uninfected macrophages to to HPV types 6, 11, 16 and 18 with 2 doses beginning between phagocytose and kill organisms released form infected mono- age 11 and 15 for girls. The quadrivalent vaccine is effective in cytes. In 1921 a vaccine for tuberculosis was developed by the preventing genital HPV infection to these types. More recently French scientists Albert Calmette and Camille Guerin (bacilli a vaccine against 9 of the most common HPV types has been Calmette Guerin, BCG) [184]. BCG is an attenuated strain of developed [157]. The vaccine is given as an intramuscular cow tuberculosis and they thought it should do for tubercu- injection. HPV is transmitted directly into the epithelium losis what cow pox did for small pox. Immunization with BCG infecting the basal (stem) cells to produce cervical cancer. induced a positive DTH reaction (tuberculin test). BCG has the The virus cannot infect intact epithelium but is able to bind capacity to limit military TB in infants, but it fails to prevent TB to basal cells through micro-abrasions or other epithelial in older age groups [184] and is pathogenic in immune com- trauma [180]. The infection then prevents maturation of the promised individuals such as HIV patients [183]. For this rea- basal cells so that continued proliferation and loss of differ- son, BCG is not recommended for use it the US, but is entiation causes cancer [181]. Since viral contact is directly to recommended in other countries. Because of the limits of the epithelium, humoral antibody cannot block viral cell inter- BCG vaccination, alternative vaccines have been prepared actions. Also, since the infection is limited to epithelial cells, and tested [185]. These include M. tuberculosis antigens in immune reactivity to the infected cells must be T-cell- subunit vaccines, adjuvants, viral vectors, including modified mediated cytotoxicity. Humoral antibody or T-cell-mediated vaccinia, adenovirus and influenza virus, inactivated mycobac- DTH are active in the dermis and not in the epithelium. terial organisms and living vaccines including modified BCG Thus, consideration should be given to intraepithelial epithe- and M. tuberculosis. None of these has yet been proved to be lial inoculation with a vaccinia virus vectored vaccine to effective in clinical trials, but if the objective is to induce induce T-CTL to infected cervical epithelium [81]. effective DTH, then the last approach (altered BCG or M. tuberculosis) will most likely be effective [186]. 2.9. Delayed type hypersensitivity (DTH) Leprosy has a similar pathogenesis as tuberculosis with bacilli infecting macrophages. The immune response in 2.9.1. Tuberculosis and leprosy leprosy determines the outcome of the infection. If there is a Tuberculosis is arguably the deadliest infection of humans; predominant antibody response there is a progressive disease over the last 200 years it has killed one billion people. It with disfiguration (Lepromatous Leprosy); if the response is currently has more victims than almost all other infectious predominantly DTH, organized granulomas will limit the pro- diseases put together [182]. The WHO estimates that in 2015 gression of the disease (Tuberculoid Leprosy) [187]. The BCG 18 S. SELL vaccine is 40% (1 dose) to 60% (2 doses) in preventing Leprosy of about 400,000 cases per year in the US. It is treated by anti- 65 [188] and is recommended by the WHO for use in countries fungal medications, such as nystatin, etc. There is no vaccine with high incidence of TB and leprosy [183]. but there is general agreement that a vaccine is needed not only because of the increased incidence, but also because of 2.9.2. Syphilis high hospitalization costs and increasing drug resistance [194]. Syphilis is a sexually transmitted disease caused by spirochete Strategies that have been used to producing a vaccine for C. Treponema pallidum. Historically the high incidence of syphilis albicans include live-attenuated strains, recombinant proteins, declined because of public health measures and the introduc- and glycoproteins, including use of water-in-oil emulsions tion of penicillin, to which T. pallidum is very sensitive. [195]. Although promising results have been obtained in Currently it is still distributed world-wide with an estimated experimental animals, no vaccine has advanced to clinical global burden of 18 million and 5.6 million new cases per year. trials. There are several major problems. First, the organism is It is very high in low income countries and in the United States extracellular, and infection does not involve binding or entry it has been sharply rising in men who have sex with men and into cells, thus many of the immune effector mechanisms in newborns of infected mothers (congenital syphilis) [189]. It applied to other infections, such as receptor blockade and T- has been estimated that congenital syphilis is responsible for CTL, are not applicable to Candida. Then, the organism transi- up to 9.5 million years of life lost [190]. The syphilis spirochete tions among yeast, pseudo-hyphal and hyphal forms, as well literally swims in the extracellular fluid of infected tissues; it as a heritable white to opaque phenotypic switch, providing a does not react with any tissue cells [191]. At the site of con- moving target for the immune response [195]. In addition, tact, about two weeks after infection, there is a firm raised normal residence in the intestine may lead to tolerance to a inflammatory reaction characterized by central necrosis, called third independent epigenetic state [195]. It appears that the a chancre. This is the result of a delayed hypersensitivity most applicable immune effector mechanism is delayed reaction characterized by lymphocytic infiltration followed by hypersensitivity (T-cell-mediated macrophage activation). As an influx of macrophages that phagocytose and destroy the with Leprosy, an antibody dominated immune response is invading T. pallidum [191,192]. This is evidence of a strong associated with progressive disease, whereas macrophage CD4 + T-cell immunity and after healing of the chancre there activation limits the disease. Thus, vaccination should be direc- is strong resistance to reinfection (chancre immunity). ted to DTH. This poses a problem for Candidiasis. For DTH the However, in about one-third of infected humans T. pallidum method of choice would be recombinant BCG as proposed for organisms have spread to other sites where they are protected a syphilis vaccine. However, the immunocompromised from immunity and cause chronic inflammation and tissue patients for whom a vaccine is most appropriate, not only damage (secondary and tertiary syphilis). Clearly, a vaccine have a limited ability to produce DTH, but also are susceptible for syphilis would be very effective in reducing the disease to BCGosis, an active infection with BCG. In any case, it does in high-risk patients [189]. There is presently no vaccine for not appear that there will be a vaccine for candidiasis in the syphilis and to the best of the writer’s knowledge, no efforts to foreseeable future. produce one [193]. Potential immunogens belong to the T. pallidum repeat (Tpr) family of proteins and adhesion proteins 2.10. Alzheimer’s disease on T. pallidum. Since the protective immune response in syphilis is delayed hypersensitivity (DTH), whatever immuno- Vaccination for Alzheimer’s disease (AD) poses unique and gen is selected it should be constructed to induce DTH [191]. challenging problems as the target is not an infectious For this goal recombinants of Bacilli–Calmette–Guerin with agent, but an autologous protein. AD is a progressive loss of DNA sequences coding Tpr or adhesion proteins is recom- memory and cognitive thinking due to the accumulation of mended [192,193]. misfolded protein breakdown products in neurons in the brain [196]. There are about 30 million people in the world with this 2.9.3. Candidiasis disease (6% of all people over 65 years of age) with an Candidiasis is a fungal infection commonly of the mouth estimated 2 million deaths per year [197]. The abnormally (Thrush) or vagina (Yeast infection), and systemically in immu- folded proteins are tau and beta protein (Aβ)[198– nocompromised individuals. Thrush refers to the gross appear- 200]. Accumulation of tau produces aggregates of neurofibril- ance of involvement of the tongue with white coating that lary tangles, whereas accumulation of small peptides Aβ resembles the breast feathers on the bird, Thrush. Although results in [198]. These accumulations are there are more than 150 types of Candida only about 20 can believed to cause loss of neural function and the symptoms cause infection, by far the most common is Candida albicans. of AD. The rational for vaccination is to prevent AD is to Individuals at high risk of infection are babies less than produce antibodies that will block the accumulation by pre- 6 months old (6%), and those receiving chemotherapy for venting the peptides from reaching the neurons. The rational cancer or with AIDS (20%). In healthy individuals, Candida is for treatment of AD is to produce antibodies (or passively part of the normal flora of skin and intestine but may become transfer antibodies) that will remove the accumulations from a localized infection of the skin, fingernails and toenails, or the affected neurons [201]. Some antibodies are not able to mucous membranes of the oral cavity or genitalia [194]. enter neurons and work in the extracellular compartment Candidemia is a blood infection that even with treatment where they may sequester tau aggregates, interfere with has a mortality rate of 50%. This condition is on the rise, assembly or promote microglial phagocytosis [200]. especially in immunocompromised patients with an incidence Antibodies that can enter neurons bind to tau aggregates EXPERT REVIEW OF VACCINES 19 within the endosome/lysosome and promote disassembly, activated the cell will be lysed and release organisms taken up leading to enhanced degradation by lysosomal enzymes; by uninfected activated macrophages that can then destroy sequester tau assemblies in the cytosol; or promote proteaso- them. This mechanism is also extremely effective against mal degradation via E3 -protein ligase binding [200]. syphilis infection, even though the organisms are extracellular. For AADvac-1 is for Tau epitope 294– Activated macrophages actively phagocytose and lyse T. palli- 305 and ACI-35 is to PSer396,404. These have proven to be dum in the interstitial fluid. safe in Phase-I clinical trials and have advanced to Phase-II trials Seven different antibodies are in Phase-I or -II trails of passive immunity [201,202]. Many variations of antibody spe- 3.1. Five-year view cificity (tau epitopes), isotope, charge, fragments, etc., have Progress can be expected in two approaches: more wide- been tested in transgenic mouse models of AD with some spread use of safe and effective vaccines, such as those for success [202–204]. As the trials are early and still in progress diphtheria, tetanus, cholera, pneumococcus, measles, varicella, there has been no evidence of efficacy in humans. Ebola virus, rotavirus, hepatitis A and B, polio, Japanese ence- A similar approach is proposed for prevention and treat- phalitis; and development of new approaches for vaccination ment of Parkinson’s disease (PD) [199]. In PD accumulation of that are directed to the appropriate immune effector mechan- aggregates of α-synuclein in neurons produces PD symptoms. ism(s). The first approach is hampered by ‘cognitive disso- Active and passive immunization is proposed to inhibit aggre- nance’. Many have religious or other irrational preconceived gation promote intracellular degradation by enhancing autop- concepts that vaccines are dangerous (cause autism, induce hagy and lysosomal degradation [205]. sterility, etc.). Even when presented with convincing data that vaccines are safe and effective, they chose for themselves and 3. Expert opinion family members not to be vaccinated. This effectively prevents heard immunity and allows diseases that should be eliminated The reduction in morbidity and mortality associated with vac- to reappear in epidemics (measles in 2019). Use of adjuvants ‘ cine-preventable diseases in the United States is one of the or conjugate vaccines may provide improved vaccines for greatest public health achievements of the first decade of the Pertussis, Anthrax; Haemophilis influenzae, Meningococcus and 21st century’ [206]. Many believe that this is an understate- S. aureus. Use of adenovirus vectors, such as Vaccinia virus, to ment. Much of the success of vaccination has been by trial and induce TCTL could be effective for Influenza, mumps virus, HIV, error. Understanding of antigens, conjugates, adjuvants, mode Hepatitis C, Chagas disease, HPV, and malaria [207]. For yellow and route of administration, and testing have progressed so fever, Dengue, West Nile virus, Typhoid, Lyme disease, that vaccines may be designed to elicit induction of the Helminths, TB, Leprosy, and Syphilis vaccines that induce immune effector mechanism (s) that will be most effective DTH may be more effective than present vaccines. The pro- against the infection. The protective and therapeutic effector blem is that a consistent, safe and effective way to induce DTH mechanisms are listed in Table 2. The first mechanism has not been identified [207]. The present state of the art ‘Inactivation of biologically active molecules’ is effective indicates that recombinant BCG may be worth trying [192], against symptoms that are caused by toxins; for example, but more effective approaches may be necessary. Adjuvants tetanus and diphtheria toxins without enlisting any other including bacterial products in water and oil emulsions are mechanisms. For pertussis and cholera toxoid vaccines are known to induce DTH in experimental animals [208], and DTH vital, but other immune effector mechanisms may be required may precede an antibody response (CD4 + T-cell priming) for full protection because inactivation of the toxin alone is when adjuvants are used [209]. Unfortunately, the DTH adju- not always up to the task of preventing the disease by itself. vants used produce local inflammatory reactions unacceptable Receptor blockade is another neutralization mechanism. This for human or even animal use. New approaches to induce DTH mechanism is the most common mechanism and is effective should be a high priority for vaccine development [210]. for infectious agents, such as viruses, that live inside cells, by preventing the infectious virus from entering the target cells. For viruses that infect epithelial cells T-cell-mediated cytotoxi- Funding city may critically enhance the therapeutic effect of vaccina- This paper was not funded. tion. For viruses that infect epithelial cells directly (papilloma, hepatitis, etc.) T-cell cytotoxicity may be the primary mechan- ism and receptor blockade a secondary mechanism. Cytolysis Declaration of interest is a primary mechanism for elimination of infected red blood cells (malaria); atopic reactions are the primary mechanism for The author has no relevant affiliations or financial involvement with any organization on entity with a financial interest in of financial conflict with expulsion of intestinal worms; and antibody-mediated com- the subject matter or materials discussed in the manuscript. This includes plement derived inflammation for bacterial infections such as employment, consultancies, honoraria, stock ownership or options, expert staphylococcus and streptococcus. T-cell-mediated activation testimony, grants or patents received of pending, or royalties. of macrophages (DTH) is the main protective mechanisms for mycobacteria that infect macrophages. Specific CD4 + T cells react with antigens on infected cells and secrete cytokines Reviewer disclosures that activate the macrophages to lyse the intracellular organ- Peer reviewers on this manuscript have no relevant financial or other isms. In situations where the infected macrophage cannot be relationships to disclose. 20 S. SELL

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