Contributed by Guest Blogger: Brittany Sider ’11
MicroRNA (miRNA) molecules, first characterized in the early 1990s, have been implicated in a variety of different biological mechanisms. It took almost a decade for researchers to detect and understand the role of miRNAs in regulation of translation. Since then, research has focused on how we can scientifically manipulate these regulating molecules to our advantage in order to further understand biological underpinnings of certain diseases, as well as potential miRNA-based therapies.
The ability of the influenza virus to undergo frequent and substantial genomic mutations forces us to continually monitor its prevalence, and modify yearly vaccines to target the prevailing viral strains. Recently, live attenuated influenza vaccines (LAIVs, e.g. FluMist) have been proven effective, and have been distributed to a large portion of the eligible population to combat the seasonal flu. These vaccines are manipulated to become much more temperature-sensitive, and therefore are only capable of replicating in temperatures found in the nose. The inability of these attenuated viruses to replicate in the respiratory tract (due to higher temperatures) allows the vaccinated individual to produce antibodies to the influenza strains in the vaccine from the infection in the nasal passage. Therefore, the individual can produce the correct immune response without the virus spreading to the respiratory tract and causing symptoms.
In 2009, a group of researchers from Mount Sinai School of Medicine found that using microRNA response elements (MREs) can supplement the effectiveness of LAIVs. In the study, the MREs for the miR-124 (neural tissue-specific) and miR-93 (a ubiquitous miRNA) were inserted into open reading frames of influenza A nucleoprotein coding regions. The investigators vaccinated mice with miR-93-seeded strains, and then inoculated them with a lethal dose of influenza A/PR/8/34 H1N1 21 days later. This resulted in 100% survival of the subjects, as well as a robust immune response. In an attempt to attribute these results to other influenza strains, the same experiment was done with H5N1 (MREs were inserted into the vaccine specific for H5N1, and methods were repeated). Subjects who had received mock vaccinations 21 days prior to being inoculated with H5N1 displayed rapid weight loss, as well as 100% mortality. On the other hand, mice that had received the MRE-containing H5N1 strain did not display any signs of disease. Furthermore, serum from these subjects exhibited neutralizing activity against the wild-type H5N1, and a wide array of antibody responses (high levels of IgM, IgG1, IgG2a and IgG2b).
The results from this study lead the researchers to believe that MRE-containing LAIVs can be used, and potentially be even more effective than currently available LAIVs in protecting against influenza A outbreaks. In addition, this technology provides the potential to control for the degree of attenuation of the vaccine by manipulating the number of MREs/miRNAs. Lastly, FluMist – although proven to be equally as effective as injected vaccines – has some age exclusions. Perhaps the addition of MREs/miRNAs could expand the target demographic of this method of vaccination.
Contributed by Guest Blogger: Joseph Zaino ‘11
Recent research has found a unique relationship between the intracellular parasite, Leishmania, and it’s corresponding Leishmania RNA virus-1 (LRV1). Ives et. al. concluded that Leishmania parasites, in the presences of LRV1, suppressed the host immune response and strengthened the pathogen’s persistence. Leishmania infects the human immune system by attacking macrophages. The parasite causes the infection known as leishmaniasis, which is typically transmitted by sand files. This is a serious infection, affecting an estimated 12 million people in the Mediterranean basin, Africa, the Middle East, Asia, Central and South America. The strain of parasite investigated by this study was mucocutaneous leishmaniasis (MCL). MCL destroys the soft tissues of the face and nasopharyngeal regions, as well as damages host immune responses.
Leishmania parasites are dependent on proinflammatory protein mediators called Toll-like receptors (TLRs). TLRs are found in intracellular vesicles of the macrophage- presumably the same vesicles that host Leishmania. Ives et. al. confirmed that TLR3-TRIF dependent pathways are essential for macrophage infection by Leishmania. The unusual part is that TLRs usually help the mammalian immune system to eliminate pathogens. Specifically, TLR-3 recognizes the double stranded RNA of many viruses that are released from dead parasites, unable to survive within their host. Observations found that between virally infected and non-virally infected Leishmania, the virally infected ones were more likely to successfully infect a host. Similarly, metastasizing parasites had greater levels of the LRVI virus than non-metastasizing parasites. The authors verified this finding by treating macrophages with purified LRVI, and observing the same phenotypic infection as the viral-infected Leishmania. Further models concluded that when TLR3 is deleted from macrophages, parasitic persistence was diminished.
This apparent mutualism seems to benefit both Leishmania and the virus by allowing a more successful rate of host infection. Many Leishmania species have lost RNAi interference pathways, allowing viruses to inhibit and replicate within them. In this case, the virally infected parasite is more persistent against macrophages, and more damaging to the mammalian immune system. Thus, it is advantageous for the parasite to coexist with the LRV1 virus. If severe MCL infections are contingent on LRV1 for infection, then future research can perhaps focus on this relationship in order to better understand and cure leishmaniasis.
Contributed by guest blogger: Nicole Engelhardt ’11
Usually when you get a vaccine it means you get a needle and a bandage. Not only that, but you get an attenuated virus. These weakened virus particles are strikingly similar to viable ones; they even infect cells. Because of their weakened state, they infect slower than natural virus particles, giving the body time to react. However, people who have weakened immune systems can still exhibit symptoms as if they were infected by the natural virus.
But a new tool may make this issue obsolete. What really matters when it comes to a vaccine is the shape of the particle, not the contents. The shape is recognized by B-cells in the body which then reproduce creating antibodies that attack all of the virus particles. However, these B-cells are very specific and very picky. Normally, it makes sense to use a weakened virus because it has the exact same shape as a normal virus and your B-cells will react to the vaccine as if it were the real thing. Is there any way, then, to produce the exact shape of the virus and therefore the correct antibodies without having the harmful side effects?
This paper explores the rotavirus particle which is the leading cause of gastroenteritis in the world. In some parts of the world, gastroenteritis can be deadly for many children. As it happens, the shape of the rotavirus particle can be mimicked almost exactly in plants. The shape of this virus is a capsid made out of proteins. First, the authors take the genes that code for the capsid proteins and insert it into the genome of the plants. Then the plants express the viral genes, creating the virus capsid proteins inside the cells of the plants. More incredible than that, these proteins self-assemble into the exact shape of the rotavirus capsid. Now you have a plant containing just the shell of the virus!
The experiments are still in their early stages, but when mice were fed these plants, the authors found they were producing the same antibodies that are produced when mice are actually infected with rotavirus. This bodes well for future research in humans. Once the antibodies are created, the severity of future infections is greatly decreased. If these transgenic plants do work, it could mean a safer and perhaps more affordable form of the vaccine that could help people the world over fight rotavirus before it can infect.
Contributed by Guest Blogger: Jack Bulat, ’11
Highly active antiretroviral therapy (HAART) has extended the quality and expectancy of life for people infected with HIV-1, but has been unsuccessful in leading to a cure for AIDS. This is because it proves ineffective at targeting the latent HIV-1 reservoir – a pool of memory CD4+ T cells in the quiescent phase of the cell cycle that harbor inactive integrated virus. Should an HIV-infected patient ever come off HAART, activation of this latent pool would cause the virus to re-emerge. Because HAART has become both expensive and toxic in the long-run, significant efforts have been directed at targeting HIV-1 latency for more effective treatment.
A considerable obstacle to studying HIV-1 latency in memory CD4+ T cells has been the lack of a latency cell model. Because only a small portion of CD4+ T cells infected with HIV-1 survive to become latently-infected memory cells, a resilient cell line mimicking latency has practical value for therapeutic screening. In a study, Yang and colleagues transduced primary CD4+ T cells with a lentiviral vector for constitutive expression of Bcl-2, an antiapoptotic signaling factor implicated in the generation and maintenance of memory CD4+ T cells. Upon confirming that the physical and biochemical properties of these Bcl-2-expressing cells are highly similar to those of freshly-isolated primary resting CD4+ T cells, they activated and infected the cells with an HIV-1 strain mutated to mitigate cytopathic effects. After establishing latency in the infected cells, the researchers screened more than 4400 drugs and natural products for the ability to activate the latent HIV-1 mutant. 5-hydroxynaphthalene-1,4-dione (5HN), a compound found in the leaves, roots, and bark of the black walnut tree, was a promising hit because it did not cause global T cell activation, which would be too dangerous for clinical use.
Despite this, it looks like 5HN will not be hitting the pharmacy shelves any time soon, since it is chemically reactive, affects several cellular proteins, and leads to the stimulation of inflammatory genes. Nevertheless, the study is significant for presenting a methodology for generating potentially useful cell lines modeling HIV-1 latency. A noteworthy criticism has been that a mutated strain, rather than wild-type virus, was used to infect the model cells. The scientists contended that the strain is suitable to study latency specifically because the genes implicated in HIV-1 activation were not modified.
Once a cell is infected, our immune system clears the infection not by targeting the virus, but by simply killing the infected cell. Antibodies are also an important part of our defense against viral infections, but their function is limited to targeting viruses floating around outside the cell. It is believed the once the virus enters the cell, it’s safe from antibodies. But a recent study suggests that this may not be true; it seems that antibodies can eliminate viruses from inside cells too. This is particularly groundbreaking because this may be a mechanism to cure infected cells, rather than just killing them.
Researchers identified a protein called TRIM21 that binds antibodies, but it was localized to the cell’s cytoplasm. Why would it be there, when antibodies are secreted? It seems that sometimes viruses can enter a cell with antibodies bound to it. While some antibodies are neutralizing, meaning that they prevent attachment to the receptor, others are not. If the virus is coated with these non-neutralizing antibodies, it can still enter, and it bring the antibodies in with it. TRIM21 recognizes these internalized antibodies, with virions attached to them, and then targets it for destruction in a cellular blender called the proteasome. In an appropriately named “fate of capsid” experiment, the researchers showed that antibody bound adenovirus capsid proteins were being degraded as soon as 2 hours after infection and that the degradation required both the proteasome and TRIM21. So the virus is being destroyed quickly, before it gets the chance to replicate.
As always, this study introduces many new questions. They used adenovirus, which causes mild respiratory infections, but does the mechanism extend to other non-enveloped viruses? Can this mechanism be used to improve existing vaccines or develop new ones? How important is this mechanism in the overall response to viral infection? Have viruses evolved mechanisms to block TRIM21?
The flu comes back year after year, and every season we get vaccinated (well, some of us anyway). Why do we need to keep getting a new shot for the flu while for others, like measles, we got way back in childhood and are done with it for the rest of our lives?
Our immune response to influenza involves production of antibodies, large proteins that specifically bind to the virus and help clear it out or neutralize it. It seems like the key to influenza immunity is neutralizing antibodies, antibodies that bind to the virion and prevent it from attaching to the host cell. You can imagine this large protein just being physically in the way, preventing the virus from binding the host receptor. The immune response that develops from natural exposure or vaccination generates neutralizing antibodies to HA, a viral envelope protein that is necessary for attachment to the host. I’ve mentioned HA in a previous post about influenza. The problem is that last season’s neutralizing antibodies dont bind to this season’s virus. Although it may be nearly identical to the virus from a past season, the new strain’s HA is slightly different, and those differences are enough to evade existing neutralizing antibodies.
Now a new approach to vaccination has shown that it may be possible to develop a vaccine that illicits broadly neutralizing antibodies, that is, antibodies that will protect against influenza strains with slightly different HAs. They used a prime/boost approach, in which a DNA vaccine was used to induce an initial response against HA, and then boosted with a regular seasonal flu vaccine. The only difference between this and what is currently done is the addition of the DNA vaccine. However the response seems quite different. Neutralizing antibodies were generated that can neutralize a variety of different influenza viruses. It seems the vaccine induced antibodies to a different part of HA. Antibodies are so specific, the dont recognize the whole HA, but rather discrete parts of it. The part recognized by these antibodies, called the stem, is highly conserved, meaning it doesnt change season to season.
This raises many interesting questions and possibilities. Could we soon have a universal vaccine that will protect us for life or at least for many years? Why did the change in vaccine regimen induce antibodies to a different part of HA? Why does the current vaccine or natural exposure fail to develop antibidies to the conserved portion of HA? Will the conserved portion of HA eventually change too, if sufficient selective pressure is applied through mass vaccination?
The 1918 influenza pandemic (the “Spanish Flu”), by some estimates, killed as many as 100 million people in a very short period of time. The 2009 “Swine Flu” pandemic didnt kill so many, but it spread rapidly and widely across the globe. Despite that difference, it turns out the two viruses responsible for these pandemics have some important similarities.
Influenza virus has a protein on the surface called hemagglutinin, or HA, which is used to attach to host cells, allowing the virus to then enter and replicate. HAs change rapidly, which is partly why influenza keeps coming back. When HA changes, your antibodies dont recognize it so well, so you get sick again. It turns out that the HAs of 2009 and 1918 are similar on both the sequence and structure level. There is a small patch on the HA protein that is 95% identical between 1918 and 2009 but only 70% identical to seasonal strains. Looking only at the 3D structure, among all influenza HAs, the 2009 HA is most similar to the 1918 HA. The 1918 and 2009 HAs also lack glycosylation at the tip, while seasonal influenza viruses HAs are sugary.
Why is that interesting? An unusual pattern was noted in the 2009 pandemic: elderly people were not as affected as younger people, the reverse of what is usually seen with influenza. It was proposed that perhaps some people still had immunity to the 1918 virus, which continued to circulate for many years after 1918, and that immunity was cross-protective. A recent study shows that this indeed seems to be the case. Mice immunized with the 1918 virus are protected against the 2009 virus. The converse is also true: if you immunize mice with the 2009 virus, they are protected against the 1918 virus. That’s pretty impressive when you consider that one season’s vaccine might not protect you from next season’s virus. It seems the immune system cant really tell the difference between these viruses. Note that it also tells us how long immunity can last! The next question is, how and why has this HA structure come back?
In 1980, smallpox was declared eradicated following an intensive global vaccination campaign. The virus, Variola, has some close relatives that can infect humans, one of which is monkeypox. Monkeypox isnt nearly the problem that smallpox was; it has a much lower mortality rate and outbreaks tend to fizzle out quickly due to poor human-to-human transmission.
However, a recent paper suggests that monkeypox infections are becoming an increasing problem. So why is it emerging now? Its a problem we’ve been anticipating, actually. Turns out that when you get the smallpox vaccine (or smallpox itself), it also protects you from monkeypox. So pre-eradication, most people were immune to monkeypox. If you met up with an infected animal, chances are you were immune and wouldn’t get infected. If you did somehow get infected, chances are most people around you were immune so you couldn’t transmit it to others. An immune host is not fertile ground for viral replication, so whenever immune hosts are encountered, the chain of viral transmission ends. In fact, a highly vaccinated population helps those few individuals that are not vaccinated by greatly limiting the potential of the virus reaching the unvaccinated (“naive”) individual. Thats called herd immunity.
Turns out herd immunity to smallpox, and therefore monkeypox, is waning. Vaccinations stopped in 1980 so anyone born after that is naive and therefore there is a major lapse in herd immunity. Risk of infection with monkeypox virus is now as much as 20 times greater than 30 years ago. Interestingly, all those old people born before 1980 who were vaccinated have a much lower risk of infection, telling us that immunity from vaccination lasts 30+ years.
So why should we worry about waning herd immunity to a rare and relatively mild disease that is hardly contagious? Well, variola and monkeypoxviruses are about 96% identical. We dont know how much monkeypox needs to mutate to become sustainable in humans or more virulent.