Discussion

Februaty 2000: (ll)S63-S73

Discussion Philip James, D.Sc., Moderator

Viral Evolution

Blumberg: I am overawed by Eigen’s approach and in- sights. I would like to touch on three aspects. First, the impact of some mutations on viral viability. In hepatitis B, a virus with one of the smallest number of base pairs, i.e., 3200, one can see a parallel structure with the even smaller hepatitis D virus, where there is overlap in their DNA. Thus, if a mutation occurs in the surface antigen-rigging frame, this induces a change in the polymerase gene with many of these types of change being lethal. If a mutation affects a vital area of the polymerase gene, then the mutant cannot persist. This is an example of the many possible constraints on the options for mutational change.

My second point relates to the origin of life. I am now the Director of NASA’s Astrobiology Institute where we are studying the origins and evolution of life on earth and indeed in the universe. Eigen made the important point that viruses can exist as crystals which can then enter cells and become biologically active. Is this the format which existed originally when conditions on the early earth were so harsh? Or was life started somewhere else in the universe and transported to earth?

Finally, we have heard of the effects of selenium deficiency and its promotion of viral mutational change. This has parallels with the nutritional toxin, aflatoxin, which interacts with hepatitis B. We always recognized that aflatoxin played a major role in inducing liver cancer, but sub- sequently we found that hepatitis B, and later hepatitis C, were required for inducing a large proportion of the liver cancer cases. Thus the risk of liver cancer is 7-8 times higher in hepatitis B carriers, whereas aflatoxin increases the risk three to fourfold. Where both risks coincide, however, the risk ratio is 60, so there is a major and poorly understood amplification. I wonder if this has real parallels with the selenium story?

Eigen: Some years ago we looked at the phylogenetic trees for the coding of Eubacteria, mitochondria, and chlo- roplasts, etc., and studied the evolution of the genetic code. We studied the evolutionary tree and the nodes where branching occurs. At each of the nodes we could look at whole families of organisms, mitochondria, etc. Our data converged nicely to show that primitive genetic codes are between 3.9 and 4.3 billion years old. The earth is about 4.7 billion years old, the big bang having oc-

curred 10-1 5 billion years ago. Cooling occurred on earth perhaps 4.4 billion years ago, so the genetic code is almost as old as the inhabitable earth, but not older. We cannot assume the code was derived from elsewhere where conditions were less favorable. So the code originated on our planet in a surprisingly short time with the genetic code of organisms being evident almost 4 billion years before humans emerged. The code probably started with the most stable interactions of bases so GGC, which yields glycine, and GCC, which yields alanine, were the first and most frequent amino acids. The evolutionary process must have had many viable mutational possibilities to allow for the complexity of adaptation to changes in the environ- ment. We did studies on this and the possible ways in which Darwinian selection might apply and found an as- tonishing speed of adaptation-within 70 minutes we had 500 generations of possible options with an in vitro viral replication system.

Lean: We think of viruses as potentially harmful but could there be protective, health-promoting viruses?

Eigen: We don’t know, but these could be produced and then retained very rapidly.

Blumberg: The study of hepatitis B virus suggests that of the 5 polymorphic loci related to the host animal’s differential response to the virus, there are loci which affect the likelihood of hepatitis carrier status. This, in turn induces a higher level of total body iron. So young people on a low iron intake may actually have been in an advan- tageous state by having better iron stores when they were infected-in the old days they did not live long enough to develop the problem of liver cancer.

James: Bud Tennant’s studies on woodchucks suggest that hepatitis carrier status is best induced by infection at a very early age. What is it about youth which affects that process?

Blumberg: In humans, as well as woodchucks, infection before birth or when very young greatly increases the risk of becoming a carrier. This could be a feature of early maturation of the liver cell or a reflection of the immune state when very young.

James: With Eigen’s evolutionary machine and liver cells or sections of different ages, could one test to see if particular viral constructs are involved in establishing carrier status at an early age and how nutrition affects the infection?

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Combs: I suppose one could use the same approach to look at the impact of HIV on immune function and on nutritional status and see how the viruses evolve and, therefore, assess the relative importance of nutritional change, of oxidative/antioxidant metabolism, and so on?

Garza: In your viral evolutionary machines, how important is the substrate that you start with in determining the outcomes of the viral evolution?

Eigen: We have not thought of doing the nutritional experiments but they could be done. It is claimed that the mutational process is random but this is not true. Selec- tive processes do allow a particular mutant to emerge so if one wanted to study the impact of nutrition, one would need to do systematic studies in experimental animal systems and not in vitro.

Hanson: The host defense system is slow to adapt although there are many more systems involved than we have been aware of. Given the huge capacity of viruses to produce new forms, how do we achieve the balance so that some viruses survive but so does the host?

Eigen: I mentioned the Sabin vaccine where there are only two mutations which distinguish it from the pathogenic wild type. Once taken by children, the virus can revert to the wild type in 1-2 days, but by then the immune system is activated. The same mutational process with HIV vaccine would not allow survival-each virus has its own rate of evolution and we have not solved the problem for HIV yet.

Goldman: I am worried that incomplete viruses may also be hazardous-they may need the presence of a complete virus to allow high replication rates.

Eigen: We have not looked at this, but we have to be careful because our understanding of viral structure does not allow us to predict their biological impact.

Glass: I was struck by the evolutionary issue relating to two single-stranded RNA viruses: the poliovirus and the Calicivirus. With polio we have three serotypes which have been around for ages so are relatively stable, whereas the Calicivirus is highly variable and there is no good immunity to it. Is this a function of the polymerases?

Eigen: The polymerases are all base adapted and single-stranded RNA viruses cannot error-correct because they do not have a reference system. We do not understand exactly but the poliovirus is having to pass through the intestine. We found that errors in the first and second codon positions are not tolerated-and you do not find them. In practice the first and second positions are en- tirely randomized, but at the protein level there is not much change so the immune system is able to cope.

Norum: Can you predict now which virus can be used to make a safe vaccine?

Eigen: No. In polio the vaccine works because the proteins do not change very much, whereas the high adapt- ability of the AIDS virus does not make me optimistic that we can find a vaccine.

Garza: I am not clear whether the likelihood of a virus tolerating a mutation as it does with HIV, but not polio, is determined by the type of protein encoded by the entire virus or relates to that specific change in the sequence.

Eigen: There is a proportionality between the number of nucleotides in the genome and the error rate. If the wild- type virus is clearly superior to the mutant, then the quasi- distribution of the viral species has a superiority two- to tenfold greater, i.e., it has a log superiority of about 1. But if one has many changes which are essentially neutral in their relative superiority, then the superiority is actually 10”. So the tolerance depends on the pressure set by the mutant which is coexistent. One needs to undertake clon- ing experiments but very little has been done with the AIDS virus.

Hanson: Let me make a similar case for the striking capacity ofthe immune system where to have T-cells which were specific for all the viral and other options would require a huge mouse of 7Og! The answer is the enormous cross-reactivity of the immune cells, so a limited number of cells cover hundreds of thousands of specificities by having responses which are almost right for the variety of virus types.

Eigen: Cesar Millstein told me that his analyses of mutational rates in immune cells is of the same order as we find in the viruses. So this is the compensatory response of the immune system. Our analysis of the mutation rate of substances that really count is 1O-Io in humans and the high mutation rates in the immune system involving the recombination of various exons means that the immune system can cope.

Glass: Have we not changed the superiority of the wild type of polio by immunizing the entire world’s population so that all the pressure now is to evolve alternatives to evade that immunity?

Eigen: I was talking about superiority in a different way and you may be correct in your concerns.

Nutritional Determinants of Viral Evolution

James: Melinda Beck, we have heard about the phenom- enal mutation rates of viruses and that selenium and vitamin E deficiency affects the outcome. What has this got to do with the selection processes?

Beck: Nutrition seems to affect the mutation or selection rate of viruses. In Cuba in the early 1990s the short- age of food and increased demand for exercise because of the collapse of the Soviet Union led to a fall in vitamin E, selenium, and carotenoid status, and the Cubans became concerned by an epidemic of neuropathy which they sought to ascribe to nutrition so that the tourists need not worry because they would continue to be well fed! It was found that 85% of patients with epidemic neuropathy had an unusual coxsackievirus, which was odd in being very slow growing and producing very small plaques. On se-

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quencing we found 95% homology with coxsackie A9 but specific mutations in the protease so the capsid region was disturbed and probably explained both the slow growth and the new pathogenic potential. The normal coxsackie A9 virus was already circulating in the Cuban population prior to the epidemic so we proposed that a mutant virus had emerged as a result of the oxidative stress in the smoking population on a poor antioxidant intake. We consider that it is the oxidative stress which was the driving force for the emergence of the pathogenic virus. So, whereas others talk of global transport, cutting rain forests, etc., I think nutrition may be an important factor in the emergence of new viruses.

Garza: Is the mutated virus dependent on a new event or the selection of part of the quasi-population of pre- existing viral forms facilitated by the antioxidant status of the individual?

Beck: I would bet on the selective pressure effect. Given the dramatic effects on T-cell function ofpoor nutrition there may also be better viral replication because of poor T-cell responses-we certainly see increased replication rates under impaired immune hnction with oxidative stress conditions in our studies and a new consensus sequence emerging more rapidly. We have done studies with MIP- 1 alpha knockout mice where the coxsackievirus only induces inflammatory myocarditis if selenium and vitamin E deficiency are also present so there are nutritional, immune, and viral factors.

ScherstCn: Could you do these studies in selectively immune deficient animals so you can identify the precise immune system that is involved, i.e., by passaging the virus through nutritionally deprived animals with or without CD4 or CD8 deficiency?

Beck: Yes, we could do that but have not done so. Arthur: How severe a selenium deficiency do you

need? We see a wide range of selenoprotein changes dependent on the deficiency level. Have you, for example, done any work on glutathione peroxidase knockout mice?

Levander: The animals are only on a selenium-deficient diet for 4 weeks so the deficiency is modest with a 40% fall in glutathione peroxidase. We have also used gold to knockout the selenoenzyme thyrodoxin reductase; by suitable gold dosing, the reductase is knocked out completely but the peroxidases persist. The ability of the benign virus to cause myocarditis does not relate to the reductase activity so we persist with our peroxidase theory.

Arthur: The changes in enzyme are analogous to those changes seen in practice in the human population, so these observations could be very relevant.

Combs: It is important to establish that it is an oxidative effect and not a specific selenium-related mechanism. In China in patients with Keshan disease, we saw in only a few hundred cases blood levels of tocopherol below 0.4 mg/dL so they were not particularly deficient. So with modest reductions in selenium, tocopherol, and ascorbic

acid intake, and with marginal levels of circulating uric acid and glutathione, you may have the circumstances for viral change which experimentally you can only induce by more intense means. Your GPXl knockout mice may be very markedly affected in oxidative terms because we find them much more oxidatively stressed than we find with general selenium deficiency. We find from knockout studies that the first class of glutathione peroxidase is most important in determining oxidative stress. When this form of GPX is removed, then selenium supplementation is without protective effects. Will Taylor has then recently looked at the genome of coxsackie B3, HIV and HIV2, measles viruses, and some other RNA viruses and found gene sequences consistent with glutathione peroxidase. He has recently expressed this sequence in E. coli and suggests that in order for these sequences to be read, there needs to be a frame shift in reading. The implication is that these RNA viruses may stimulate the cell to produce GPX. This would presumably reduce oxidative stress and thereby reduce not only its own enzyme transcription but that of the virus so there could be down-regulation, which might allow the virus to hide away inside the cell and away from immune attack.

Beck: We have also looked at iron as a pro-oxidative stimulator and we see the phenotypic conversion to the virulent virus but we still have to do the sequencing. We are also going to look at vitamin C-deficient mice and assess whether polio and flu viruses change faster in vitamin C deficiency.

Meydani: Could we consider the elderly who have an increase in oxidative stress and an impairment of immune function? They are well recognized to be susceptible to infections-particularly respiratory. If one takes older mice, with and without supplements of vitamin E in high doses, then on exposure to the flu virus, the vitamin E- supplemented animals show a l O3 reduction in viral load; in younger animals the reduction is only tenfold. Inter- feron y production may be related but cannot explain the very early differences in viral load with and without vitamin E.

Beck: There are seven nucleotide sequence differences between the avirulent and virulent virus-once the changes in the genome have occurred, it does not matter what you are eating. We are selecting experimentally for the most virulent strain of virus by taking tissues out for study at the time of the greatest pathological effects. The way the mutational/selection pressures go seem to pro- mote virulence. On repeated experiments we always get a conversion to virulence.

Eigen: We did experiments and found the emergence of the same mutant from four different viral origins so we are dealing with some unknown determining factors.

Garza: I am confused by the concern: if we have a population of viruses with a variety of sequences and through antioxidant stress one particular virus among the


family of viruses emerges under particular ecological Eigen: Why questions are always dangerous! These conditions, then why should we be surprised on finding the same consensus collection under similar ecological conditions? The same subspecies is simply going to emerge because they survive much better so the net effect is not random at all.

Norum: You have described a general infection and a general oxidative stress. Could this also apply to a local infection and a local oxidative stress? Could that be re- sponsible for local changes, e.g., to produce a cancer?

Beck: Yes. There is a host of local events in the mi- croenvironment which could be important.

Blumberg: Has anybody compared the virus in liver cancer patients where there is hepatitis B carrier status with and without aflatoxin exposure. This type of experi- ment has broader implications. In the natural history of viral infections not everyone who is a chronic carrier de- velops cancer, whether one is considering papilloma virus and cervical cancer, HDL, and HDL,, and human T-cell leukemia viruses, nasopharyngeal cancer, and the Epstein- Barr virus-in these viral conditions perhaps there is some environmental effect which induces mutational change and the second event, i.e., cancer. If so, this has real public health significance.

Levander: It was shown years ago that selenium could protect against mouse mammillary tumor virus; at that time there was skepticism but we should look again at this issue.

Rosenberg: Could we consider the therapeutic aspects in viral infections? In the Cuban model of neuropathy there seemed to be a temporal response to the use of vitamin supplements-were the B vitamins involved and can nutrition modulate the effects once there has been a viral mutational shift?

Beck: We do not understand in Cuba what happens when the mutational shift in the virus affecting one pa- tient is passed to another. Unlike the usual coxsackievirus, which has four cleaved proteins making up the outer capsid, this mutant has one long polyprotein. The Cuban scientists suspect they are observing an atypically per- sistent enterovirus in the nervous system, then the ex- pression of this unusual virus is amplified by a nutritional deficiency through, perhaps, an impact on the immune system.

Keusch: In the conversion to virulence all the other interactions which do not affect virulence will be missed. Considering the variability of HIV and what we know about the coreceptor and conformational requirements to acti- vate a primary and secondary receptor, there has to be a limited number of changes that are tolerated and still allow the virus to be productive. So why is it not possible to find the degree of constancy in viral selection which will, in turn allow the development of a vaccine and an immune response?

constant regions are too short-if they were longer one might be able to develop an immune response, but the regions are also probably too widely distributed and the intermediate regions must allow mutations which permit escape mutants but this is just a guess.

Beck: We have a system where the virus goes first to the liver and then to the heart in a sequential process over 10 days so by studying the time-dependent changes in viral sequence we may be able to get at that question.

Combs: Are there other examples of nutritionally induced shifts in viral effects other than selenium and has selenium been looked at in the liver cancer cases in regions of China with a low selenium status? And has anybody looked at other models where there is a compro- mised immunity?

Levander: Tom London is assessing liver cancer in both China and in the Senegalese military, where there is a lower frequency of liver cancer despite high hepatitis carrier rates. They have found that aflatoxin is much lower in Senegal and there also appears to be less viral replication.

James: Colin Campbell in Cornell believes that aflatoxin has an impact if the protein intake is adequate but not when protein intakes are low.

Scrimshaw: Many Asian papers 20 years ago showed greater symptomatology from aflatoxin poisoning if protein intakes were low and we did a large study in Thailand which failed to suggest a relationship between aflatoxin and liver cancer.

Garza: Colin Campbell's work is somewhat different in that it relates to the effect of high protein intakes in lowering the LD,, dose of aflatoxin needed to induce liver cancer.

Combs: Campbell would consider that protein deficiency is linked to the acute toxicity of aflatoxin but higher intakes shift the impact of aflatoxin to carcinogenicity.

Nutrition and Immune Responses

Schostein: We should keep in mind that our defense against viruses is very dependent on cell-mediated immunity and the production of interferon y is very sensitive to the action of free radicals. We also known that n-6 fatty acids are very potent inhibitors of interferon y production mediated through cyclic AMP. These effects are powerful stimulators of the TH2 type of immune response but strongly inhibitory for TH1 immune effects. We do not know the effects of n-3 fatty acids although they do enhance the interferon y response of mice infected with List- eria.

Hanson: n-3 Fatty acids increase leptin levels, which will increase TH1 responses and increase the production of interferon y.

James: There has been a remarkable shift in the n-6/

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n-3 fatty acid ratio in the human diet over the last 50 years as n-6 intakes have been promoted to help prevent heart disease, while the n-3 content of plants was reduced to help limit the problems of rancidity. Perhaps these shifts have had substantial effects on immune responses to viral infections?

Meydani: The effects of n-3 fatty acids are complicated: they both decrease prostaglandin production and increase oxidative stress so with some infections there could be a promotion of the virus. Studies with flu suggest n-3 fatty acids reduce host resistance but the impact seems to be virus specific.

West: n-3 Fatty acids in mice and humans give oppo- site effects in terms of interferon y responses.

Blumberg: The iron impact on hepatic B carriers needs to be considered because in Korea there was a strong correlation between high iron levels in the liver of patients with hepatitis B and the likelihood of their developing liver cancer. There are similar findings in mice. So perhaps we had better be careful about iron fortification of food.

Fetal Immunity

Antipatis: We have studied experimentally the effect of vitamin A deficiency during pregnancy and find a specific immunological response in the placenta with neutrophils producing TNFa. This could impair pregnancy but TNFa also induces leptin. We have also shown that vitamin A deficiency affects fetal lung development, probably per- manently, so this may explain the failure of vitamin A supplements to reverse the respiratory disease problem in vitamin A-deficient societies-the children may have been born with a hnctional deficit of the respiratory immune system.

Hanson: This is important with leptin potentially con- tributing to the loss of appetite; your observations may also relate to the recognized impaired fertility of vitamin A-deficient patients.

Stoll: The best model for malnutrition is the low birth weight baby, which in developing countries reflects intrauterine growth retardation. But in the U.S. these babies are premature and in these chronic lung disease is common. Postnatal supplements lead to a modest improvement independent of infection rates.

Goldman: The early programming ofthe immune system is important with crucial windows of opportunity for appropriate immune development. We need to understand this so immunization programs take the appropriate timing into account.

Hanson: The immunological control of pregnancy is now being revealed. Several cytokines control the growth of the trophoblast and the development of the placenta. Implantation also requires TNFa production but this then needs to be limited. Placental growth is cytokine controlled

and includes the impact of insulin growth factor I . Ifpar- ents are antigenically very different, the placenta is larger and we seem to select our mates by processes which include a nasal sensitivity to smells linked to our HLA class! In intrauterine growth retardation there is a deficiency of the mRNA of interleukin IL,, within the placenta. This probably signifies that this IL,, is failing to depress the THI side of the immune system. We also know that fat feeding to the mother enhances leptin levels, which in turn promotes immunological development.

Viral Uptake and Processing

Glass: I am far from an expert in this field, but if one does have a protein or proteasome within the gut lumen then it is surprising how those molecules seem able to translo- cate across the epithelial membrane. Examples include the bacterial toxins which, once secreted by the bacteria, bind to the gut epithelium by surface catalytic subunits and then are translocated into the cytoplasm where it modifies ribosomal proteins. If this subunit could be a component of luminal viral proteasomes, then we need to consider their potential physiological significance.

James: There are now many studies in the Third World demonstrating increased intestinal permeability to a whole variety of large molecules.

Meydani: The nutritional world has tended to ignore the impact of nutrition on the gut-associated immune tissues (GALT). The function and control of GALT is very different from systemic immune responses and a crucial first line of defense. The effectiveness of oral vaccines depends on the appropriate GALT response.

Hanson: Our understanding is improving. It appears that there are intestinal surface structures which aid the transfer of antigens to the underlying dendritic cells which are layered beneath the basement membrane of the mucosal surface cells. The uptake depends on the formation of epithelial vesicular exosomes containing HLA, which are then transferred to the dendritic cells, which then respond by maturing and migrating to local sites for antibody production. There is some control system determining the switching of dendritic cells so blood-derived HLA and associated peptides seem to induce tolerance-a rec- ognition ofhost factors, whereas the Peyer’s patches with their GALT respond to incoming luminal antigens from bacteria and viruses. The responses include the genera- tion of secretory IgA, which accounts for 80% of antibody production.

In a vitamin A-deficient individual there may only be 10% of the normal IgA response. Yet, deficient individuals still manage to cope if well fed so there are many other mechanisms. Whereas vitamin A deficiency enhances some T-cell-mediated responses, there is a reduction in natural killer cell number and activity so the multiple responses to a particular nutrient can be very complicated


and affect the response to viruses. Karre: The immune response to viruses is depen-

dent on the organ involved. Natural killer cells are very effective in protecting against the early impact of cytome- galovirus. Knockout mice experiments show that the natural killer cells in the liver produce interferon y, which completely controls viral replication, whereas in the spleen the natural killer cells themselves deal with the virus. Thus, if nutrition induces interferon y deficiency, then the animals will rapidly die from a viral infection.

Keusch: Do we know of circumstances where, as in some parasitic diseases, a viral infection can have a reduced impact when the body is malnourished?

Tompkins: It is difficult to assess this because marginal malnutrition is so frequently associated with poverty and exposure to a greater number of infections. There is evidence that excess oral iron is detrimental to individuals with malaria but I do not know if iron’s pro-oxidative role can enhance viral replication or damage.

Weinberg: There is preliminary epidemiological data that hepatitis C is worse in people who are iron overloaded and HIV infections progress faster in Italian and French patients with high body iron loads as a result of thalas- semia. Iron chelation seemed to help in reducing the progress of the viral infection.

Blumberg: One study suggests that iron overload increases the risk of becoming a chronic hepatitis B carrier. The progression to cancer also seems to relate to high body iron levels.

West: We may need to distinguish between the effects of oral and parenteral iron.

Viral Load

James: Could we improve the interpretation of nutritional interactions by systematically obtaining some measure or index of viral load?

Glass: With many respiratory and enteric viral infections, the severity of the disease and the viral load go hand in hand so monitoring viral load may be helpful in HIV but not with many other viruses. The big question is how nutrition affects viral proliferation; in rotavirus infection, 1 in 150 Bangladeshi children dies compared with 1 in 150,000 American children. Both groups excrete 10I2 rotaviruses when acutely infected, so why the difference?

Tomkins: With an equivalent viral load the difference must be in the tissue inflammatory response. With HIV, however, if we boost a mother’s nutrition, do we then boost the viral load and increase the risk of maternal transmission-an important practical issue.

West: Studies have looked at this problem and have not shown an increase in viral load when vitamin A has been given-vitamin A actually reduces maternal transmission in most studies but other nutritional effects may be different.

James: Roger Glass has dismissed the viral load issue, but African babies have a totally different clinical picture with measles than European children. Is there no relationship to viral load but just the tissue response?

Glass: It could be measured: one can have remarkable effects on mortality by prior immunization even in malnourished populations.

Stranneglrd: The viral load question is interesting. Essentially, we know little except in HIV and hepatitis C. Plasma levels of HIV RNA are used as an index of viral load and, independent of the viral subtype, one finds no difference in load in Swedish and African patients; if one looks at African immigrants in Sweden they have the same natural course of the disease, but under African conditions they deteriorate much more rapidly.

James: If nutrition is then crucial to the response, we need to recognize that our recent analyses of adult malnutrition in different parts of the world reveal a substantial problem. In rural India, for example, half the adults are underweight as judged by a body mass index of <18.5 and have associated increased morbidity. This morbidity in underweight adults is replicated in African and Brazilian studies. Furthermore, in Indian studies simple classic mea- surements of both cell-mediated and T-cell immune responses were reduced and we have found (with WHO and Ferro-Luzzi) multiple vitamin deficiencies. The immune status improves with micronutrient supplements so we may have seriously underestimated the importance of nutrition in the capacity of adults to resist viral infections.

Stranneglrd: The TNFa responses in the HIV patients in Ethiopia are five times higher than in our Swedish patients. I do not know whether this indicates coexisting infections or some nutritional effects, but it may explain why HIV induces the “slim disease” in Africa.

Keusch: We looked at a group of HIV patients in Kinshasa who had remained relatively asymptomatic for 5-7 years without therapy. We found their proinflammatory cytokine levels to be sky high but when we also measured the anticytokine regulators, e.g., the soluble TNF receptor, they were also extremely high, so a balance had been achieved. This shows the importance of assessing the responses as well as the viral and cytokine loads. The responses in nutritional terms also need to recognize that the acute phase response (APR) to infection induces selective channelling of nutrients to specific tissues-the plasma nutrients themselves are doing nothing.

Norum: In addition to the impact on circulating vitamin A levels of a fall in retinol binding protein in the acute phase response, we also have to consider the processing of oral vitamin A. The chylomicron remnants with their vitamin A will target the liver and bone marrow first so we may have a distorted picture if we consider only events in the fasting stage-there is a diurnal variation in vitamin A processing.

Hanson: Recent studies have shown that a huge num-


ber of lymphocytes are produced daily to try to cope with the HIV attack, but the lymphocytes are rapidly destroyed by a necrotic rather than an apoptotic process, which means that a substantial mass of nucleotides will need to be synthesized each day. Lymphocytes, like the intestinal mucosa with its fast turnover, cannot generate enough nucleic acids de novo so I wonder whether we should be thinking of nucleotide feeding in viral infections.

Scrimshaw: The body’s response to infection can be much greater than we expect-in studies with the 17D strain of yellow fever we still find a very clear negative nitrogen balance without any associated temperature rise or symptoms. Similarly, studies with volunteers given Q fever showed that a subgroup had no symptoms at all but they still developed a negative N balance, which lasted more than 3 weeks. Some immunization studies in children have also documented their subsequent loss of weight and failure to grow. The impact of immunization or infection will vary, of course, in part due to the frequency of antigen presentation-in Boston children an infection has no long-term impact on growth, whereas Mata’s studies in Guatemala show that infection rates determine the ex- tent to which a child’s growth deviates from the local av- erage growth rate.

Hanson: The interindividual differences may relate to the gene polymorphism of both the inflammatory and im- munosuppressive cytokines. If one has the allele which induces a high TNFa response and the individual also has the allele linked to a low production of the more immu- nosuppressive interleukin IL,,, then symptoms may be marked, whereas the reverse combination may be linked to few symptoms.

Nutrients and Viral Transmission

Glass: We need to recognize that in practical terms we may achieve huge medical and societal gains without understanding the precise mechanism involved. Thus, we have eradicated smallpox and perhaps polio without having any detailed understanding of the molecular basis for the infective process. Similarly, in my own field of rotaviruses, there is already a licensed vaccine with more being developed, yet we have little understanding of the immune process involved in combating rotaviruses.

Ploegh: That is a valid point but by looking at the smallpox and polioviruses we may uncover new therapeutic possibilities-we may need to learn how to turn on receptors, what type of strategies viruses use to evade capture, or how to maintain the proper redox potential within the endoplasmic reticulum’s lumen so that proper protein folding is maintained. All these openings will come from further study.

Keusch: I would like to discuss Glass’s Luddite approach! It is true that the measles vaccine was developed empirically but there were various problems. Whereas in

well-nourished individuals exposed to appropriately timed vaccine there is the development of protective immunity, studies in West Africa showed early on that a large dose of vaccine had a profound effect in suppressing immunity and increasing death rates after immunization. We now realize that the viral vaccine, like the wild type, attaches to a receptor which is part of the complement activation regulation gene complex on CD46, that the virus induces apoptosis in lymphocytes with effects on interleukin IL,,, a shift from T-cell TH1 to TH2 and a variety of other immunological effects with the vaccine producing more modest changes than the measles virus itself. So we are begin- ning to understand the interactions between the virus, a viral vaccine, the viral load, and the nutritional state of the recipient, which has profound public health implications.

Childhood Transmission of HIV

Weinberg: I would reinforce Professor Peckham’s empha- sis on the burden of childhood infection especially if one considers Africa and Asia rather than the U.S. or Europe. She has well described the three phases of transmission of infection from mother to child. It is difficult when at- tempting to identify nutritional modulators of this transmission to take account of all the confounders-degrees of anemia, presence of malaria, degree of chorioendo- metriosis, or other sexually transmitted disease in the mother. I am interested in the iron status of the mother because there is some experimental and clinical evidence that iron status plays a role in the advancement of the disease. The iron status may affect the mother’s viral load and therefore the risk of transmission to the child. The recent short-term therapy studies with azothiaprine (AZT) are interesting because they do suggest a substantial benefit from short-term dosages given in late pregnancy and labor but continuing to breast-feed does seem to increase the transmission risk.

Tomkins: It seems to me that Asia will completely outstrip Africa in terms of the burden of childhood HIV infection. We have difficulty monitoring the HIV transmission in babies because of the use only of the ELISA test, which picks up the antibody transmitted from the mother to the child. So a child may remain ELISA-positive for up to 18 months postdelivery, even when without viral infection. There is no technological capacity in poor countries to diagnose an infection using DNA PCR techniques so we need to develop cheaper and rapid automated facili- ties for DNA PCR if we are to make a proper assessment. The practical importance of this is immense since an infected mother may only transmit to a quarter of her children so we may expose the other three quarters to severe risk if we manipulate feeding regimes, etc., unnecessarily.

My second point is that many of these intervention studies have been very short term, lasting 3-6 months only. For public health policy purposes, we need to follow

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children for much longer. I would also emphasize the need for caution when

interpreting low vitamin A levels in association with HIV. We know that the plasma vitamin A level is dependent on the level of retinol binding protein (RBP) so the illness associated with HIV will drop the RBP and therefore the vitamin A level independently of vitamin A status so we need intervention studies with vitamin A before we are confident about its role.

My next point relates to recent evidence of the presence of substantial mastitis in 20% of lactating women in Tanzania, Bangladesh, and South Africa. This is shown by their raised sodium and potassium levels in their breast milk. This is associated with a high viral load. These women may be particularly sensitive to high viral replication for genetic or nutritional reasons. Recently it has been shown that women supplemented with sunflower oil as opposed to palm oil or simply rice have a marked reduction in both viral load and mastitis. We may need to take nutritional effects, therefore, into account in terms of viral metabolism itself and the impact on the immune system. New data from South Afnca now demonstrate a lower perinatal transmission risk in exclusively breast-fed babies rather than those on mixed feeding. This mixed feeding seems to be associated with mucosal damage as assessed by the intestinal permeability to lactulose and mannitol. This, if confirmed, could have profound implications for poor com- munities because absolutely exclusive breast-feeding may not only minimize the risk of developing substantial mastitis but also reduce the impact of dietary and bacterial antigens on the baby’s gut and therefore on its handling of any viral load.

Now to the practicalities of breast-feeding in poor countries: there is currently complete confusion in Afri- can antenatal and maternal and child health clinics on what to recommend. It is exceptionally difficult for a woman to assess the risks and benefits. In practice, a number of women in Tanzania are using cow’s milk from birth. They may dilute an already diluted cow’s milk preparation and increase, not reduce, the hazards. Next is the problem of wet nurses and the confusion in the use of breast milk banks. In Brazil, some hospitals continue to have milk banks, whereas others have shut them down. Many poor women depend on wet nursing to allow them to go to work but the uncertainty of the HIV status of wet nurses means that this practice is no longer recommended.

From these new findings, one could theorize that the best option in poor countries is to breast-feed exclusively for 3-4 months and then stop, but this would be culturally traumatic-absolutely exclusive breast-feeding is very unusual in any population group because it signifies the exclusion of all additional water and juices so the breastfeeding is totally exclusive.

Finally, let us recognize the impact ofHIV infection in childhood. Adult physicians coping with the HIV epidemic may dismiss the problem but it has transformed the problems of childhood in Africa and is reversing all the gains from 20 years of effort on vaccination and other measures to reduce childhood mortality. Twenty years ago, Tanza- nia had an infant mortality rate of 100, which was reduced to about 60 per 1000 live births. Now we are in reverse and the death rates over the last 2 years are back to 100/120. We therefore do need appropriate and practical strategies for coping with HIV transmission to infants.

Goldman: When we consider these broader issues we may gain some insight by looking at the detailed patho- genesis of this strange infection. We knew that HIV binds to a major epitope on the helper T-cell CD4 but now we recognize a coreceptor on both T-cells and on macrophages which allows the virus to enter and intracellularly proliferate. Soon a number of cells are activated to proliferate but a series of other activated pathways lead to pro- grammed cell death, which leads to very deleterious effects on the immune system with dysregulation and im- munodeficiency.

We also need to consider natural selection processes as well as molecular mechanisms. We need to recognize that current populations are far more mobile than in Pale- olithic times and the deadly part of this infection is the long latent period between people becoming infectious and then becoming sick. The third problem relates to the specific attack on the very system which normally com- bats infection. This is the backdrop to the HIV and breast- feeding problem. Lars Hanson has highlighted the factors in breast-feeding which have the potential for long-term protection. During the colostral phase of lactation, a host of cells is transferred from the breast-some ofthese cells are found only in the early phase in decreasing order: the neutrophils, macrophages, CD4-positive T-cells, B cells, and natural killer cells. All these cells are activated by HIV and then encounter the saliva and other fluids in the body. Samuel Barron from my institute has pointed out that infected T-cells or macrophages, when encountering the hypotonic nature of saliva, burst to release the virus which can then latch onto further receptors on other cells which allow the infection to continue. Infected T-cells transferred into human milk with its relatively high osmolarity remain intact so they could evade the modest amount of saliva and be transferred as intact cells into the stomach. We are not, however, clear how the virus gets into milk. If virus moves across an epithelium then, if there is dimeric IgA present, the virus can be neutralized. However, the dimeric IgA could also enhance the transfer of virus to the luminal side: this needs to be looked at in relation to a number of viruses. There is no direct evidence of viral attachment to the intestinal epithelium but there is some evidence of the


absorption of breast milk cells through the baby’s intestine into its systemic circulation. This is found in experimental animals but I know of no comparable data in humans. So memory T-cells from the mother may transfer to the baby. We still have not, however, established the full sequence so we do not know whether this is an HIV transmission route.

Viral-Nutrient Interactions

James: We need to consider whether there are common pathways for viral processing which relate to certain nutrients and/or whether the biology of different viruses involves selective nutrient interactions.

Meydani: If we consider the nutrient responses to viral infection such as the fall in the vitamin E and glutathione levels, do these changes have anything to do with a common mechanism, e.g., proteolytic activity? Some proteolytic enzymes are very sensitive to the redox state of the cells, so is the virus using a route of attack on antioxidants to overcome a defense mechanism?

Ploegh: I do not know. A virus will turn off host protein synthesis and regear the process to producing a few viral patterns. This will alter optimum folding. I gather from discussions with Blumberg that there is a glucosi- dase inhibitor which blocks the biogenesis of viruses, but these same agents interfere with the normal physiological cycle of glycoprotein folding which involves deglucosylation and reglucosylation as part of the proper folding process. It then transpires that the viral glycopro- teins are far more sensitive than the normal cellular processes to these inhibitors. So one can make cell lines that are deficient in the chaperone that controls this reaction but the cells are still viable, whereas the virus is much more sensitive to this mechanism. I conclude that individual viruses may be very sensitive because a small set of proteins has to be made by that one cell. How this relates to antioxidant sensitivities, I do not know.

Blumberg: This glucosylation process is interesting-hepatic B virus has three sugar binding sites on its surface, whereas the HIV has many more. So an interfer- ence in glucosylation will have a profound effect on the tertiary structures of virus. This could have useful therapeutic implications.

Lonnroth: In our research, discussed by Lars Hanson, we are looking at an antisecretory factor produced by the mammary gland as well as the intestine, pituitary, and placenta. Recently it has been shown to bind ubiquitine and polyubiquitine, thereby regulating proteasomes and their breakdown. The binding is like that of an antibody rather than covalently. The immediate effect of this factor is to bind cholera toxin but then the factor seems to be taken up by the gut where it seems to induce protein breakdown. So we need to be clear how nutritional factors af-

fect the uptake of viruses from the gut and the gut’s capacity to cope with a viral load.

The Impact of Micronutrient Deficiencies

Zinc Tomkins: Cells and tissue responses to viral infection are very different if there is accompanying micronutrient deficiency. Intestinal intercellular junctions start to fall part so one sees a much greater inflammatory and cytokine response, e.g., in zinc deficiency. Too many studies, however, have given megadoses of zinc, selenium, vitamin A, or other nutrients with no analyses of how this impacts on the copy number of the viruses such as HIV or rotavirus. Large doses of zinc in severely malnourished Bangladeshi children have increased mortality, the problem of reduced copper availability being neglected so we need to look at the physiological and functional importance in women and children of a range of graded doses of micronutrients.

West: Once one does see great benefit, e.g., with zinc improving appetite and childhood growth rates and reduced respiratory and diarrheal disease in Ethiopia, then it is very difficult to justify further studies with a placebo group so that one can look at the more detailed viral-nutrient interactions.

Edqvist: In our extensive studies on the increase in piglet diarrhea and mortality following the removal of dietary growth promoters, we found that the widespread use of zinc oxide supplementation seemed to maintain a diversity of intestinal bacteria and reduced diarrhea and mortality.

Arthur: In pigs exposed to endotoxin, which induced a fall in plasma zinc, attempts to infuse zinc to improve the plasma level killed the animals. This re-emphasizes the need to be cautious when interpreting the significance of plasma levels and the dangers of giving intravenous minerals in infected animals.

Glass: We are now involved with zinc, which is a potent adjuvant or additive in vaccine work. I agree we are worried about giving live oral vaccines to children in developing countries. One way to improve the immunoge- nicity of viruses is to give it with zinc. There was a lovely study in India where the giving of zinc regularly to children decreased the incidence and severity of diarrheal illnesses in the first 2 years of life.

James: Surely that just means that ifthe Indian child’s intestine is damaged and fecal zinc losses are high, the child needs to recover by turning over more gut protein. The child can then use the extra zinc to recover normal gut processes? It may not therefore be a selective viral effect but a generic nutritional problem?

Glass: Viruses may add to the problem of damage or zinc may interact with the viral infection itself and cer-


tainly affects the immune system as such.

Vitamin A Hanson: We know that in experimental vitamin A deficiency there is an overgrowth of enterobacteria and once small intestinal levels reach 1 08/g, the risk of septicemia increases markedly. So the mortality, particularly from chronic rather than acute diarrhea in vitamin A-deficient subjects may be explained through this mechanism.

Bruce: In Nordic countries we have had a policy of using cod liver oil or in Sweden fortification with syn- thetic retinol and vitamin D. There is now a plan to remove these-do you think this is a wise policy?

Meydani: In a recent review of vitamin A and its impact on infectious diseases it would seem, as with other nutrients, that the impact of fortification or supplements depends on whether or not the population is deficient in the first place.

West: We should remember that in Europe vitaminA deficiency and even xerophthalmia was found after World War I, in Denmark for example, so we need to recognize that our fortification and supplementary policies may be maintaining the vitamin A status of the population and I believe the policy should be maintained in Europe-there is no evidence that it is doing harm.

Scrimshaw: The impact of these nutrients on the response to infection can be odd: zinc, iron, and vitamin A can be shown experimentally to alter immune function, but if you look at vitamin A community intervention studies, one can see in the majority of studies there is a fall in mortality but without inducing a fall in morbidity. The effect of vitamin A is therefore an enigma.

Tomkins: Most of the fall in mortality reflects a reduction in the number of children admitted to clinics or hospitals with severe disease. We do not understand how micronutrient supplements change the viral pattern of infection.

West: Even if morbidity rate does not seem affected, vitamin A can reduce the duration of diarrhea or pneumo- nia, e.g., after 10 days illness, duration is reduced by 60%. Presumably this does reflect a better immune response. This of course is not dealing with a true deficiency state.

Iron Issues Combs: We need to distinguish the impact of micronutrients in deficient and nondeficient populations. Thus selenium supplements can reduce cancer incidence in a nondeficient population. We must also take account of bioavailability. Iron is the fourth most prevalent element in the earth’s crust, yet 40% of the world’s women are anemic. Instead of just assessing dietary deficiency, we should consider the potential gains from reducing the iron and zinc binder in the diet, phytate.

Blumberg: We also need to recognize the polymorphism of different genotypes affecting nutrients, e.g., iron

utilization. There is increasing evidence of population differences in the prevalence of different genotypes so the impact of micronutrients may also vary in some studies for this reason.

Weinberg: The whole population may be parasitized or infected and then one may have a further problem in calculating the response.

Selenium and Other Minerals Levander: We have potentiated the virulence of our coxsackievirus by adding arsenic or mercury to the animal diets before exposure to the virus. We considered this as an antiselenium effect but this still needs to be demon- strated.

Scrimshaw: We should not imagine that single nutrients influence a particular virus in a selective way because there are many studies showing nutrient interactions. Thus Martorell’s studies in Guatemala and a recent study in Venezuela show that vitamin A supplements lead to an improvement in iron status. We may therefore have to think in terms of testing the effect of combined micronutrient supplements and choosing the right amount with care so that we get the maximum public health impact and minimize the problem of subsections of society having a deleterious response to excess micronutrients.

Keusch: Agreed, but we see quite selective effects of iron on specific bacteria so there may be distinctive effects.

General Public Health Issues Scrimshaw: I enjoyed Dr. Brundtland’s superb and com- prehensive presentation-we have come a long way since our original WHO monograph called to public attention the synergistic interactions between malnutrition and infection. At that stage there was a great deal of clinical and epidemiological evidence but very little mechanistic understanding. In our recent multiagency meeting on iron deficiency, a case was made when considering malaria and anemia to ensure that, while malaria itself must be tackled directly, there is no contraindication to iron supplementation to combat iron deficiency.

Brundtland: Agreed. In the Roll-back Malaria Pro- gram for many countries, but especially Africa, we are try- ing to build a social movement linked to health sector development which does not consider malaria as an iso- lated vertical initiative in society but an integrated scheme which should include programs for preventing iron deficiency. In the EMRO region last fall I saw iron-fortified bread being used for their prevalent problem of iron deficiency, but clearly many strategies are available for different countries to cope with improving the iron status of adults as well as children.

Garza: There seems to be a problem in persuading policy makers of the value of preventive strategies because their effectiveness means that diseases are less


apparent! Thus, in the U.S. 14% of GNP goes on health care but less than 1% on health promotion and disease prevention. How do we change policy makers’ motivation so that money is used more effectively?

Brundtland: I recognize the problem and believe we have to present evidence repeatedly not only to policy makers but to NGOs and the public. We then need to chal- lenge, for example, the U.S. about the contrast in medical spending with the millions who live in poverty with basic deficiencies which can readily be rectified. In our next report on Health Systems we will link economic analyses to public health and provide health indices of effective spending which will reveal that with a spend of 6 8 % GNP one can achieve good health indicators.

West: In relation to your comment on vitamin A im- provements, there have been two impediments to progress. The first is the insistence by some that vitamin A capsule programs should be temporary and not specified as along- term strategy, despite their effective use in Bangladesh for almost a quarter of a century. The second problem relates to the need to choose particular foods with a better bioavailability of their provitamin A content rather than simply advocating dark green leafy vegetables, which have poor bioavailability.

Brundtland: Many times I have seen scientific wrangles delaying the implementation of any policy at all. For example, in Norway there was an academic dispute as to how best to provide fluoride to help prevent tooth de- cay-whether in water, milk, or by other means. After 25 years of inaction we finally gave children fluoride tablets to suck! I have now become far more interested in action than in academic disputes.

Tomkins: I am delighted to see WHO’S Director Gen- eral giving such a high profile to nutrition. Can you see how to transform the vision of other agencies, for example, in terms of education in poorer countries so that these agencies incorporate the value of nutrition into their programs and do not simply regard WHO as the back-up technical agency?

Brundtland: We have to talk as leaders of agencies with each other and try to complement and not duplicate each other’s mandates. We must also ensure governments get the same message so that there is a single policy and not one from UNICEF, another from WHO, a further one from the World Bank, etc. The same problem arises at a national level where Prime Ministers have to ensure that at regional and grassroots levels Ministries of Health and Agriculture have only one coherent policy. In a UN con- text we need to develop new strategies for ensuring co- herence.

Garza: We have had an erudite discussion about the mechanisms of viral-nutrient interactions but we must rec-

ognize that we need to push ahead with public health strategies even if we do not know all the mechanisms and interactions.

Scrimshaw: Agreed, vitamin A deficiency we know affects perhaps 200 million in up to 40 countries, whereas iron deficiency handicaps up to 2 billion in 140 countries. It seems clear that iron supplementation programs lead to a fall in both respiratory and diarrheal disease.

Keusch: I would emphasize that the global burden of viral diseases is not going to disappear. It is clear that malnutrition of one form or another has an adverse impact on a population’s response to infections. The global impact of viral disease will therefore depend on what we do about the world’s nutrition over the next 20 years. This will depend on how we cope with the escalating population in the Third World.

Tomkins: Ifwe look at the global change in micronutrient availability, recurrent flooding has led to massive reductions in the micronutrient content of the soil and of food. We can expect iodine and zinc deficiencies will increase. Predictions in Africa suggest that marked reductions in the micronutrient content of the soil are occur- ring. We need to consider what technologies, including genetically modified plants, we could use to combat these problems.

Glass: The enteric, respiratory, and hepatitis viruses probably account for a third of the 12 million deaths in children under the age of 5 years.

Pelletier: In our analyses of children’s weight for age as an indicator of malnutrition, we have found in re- viewing the HIV studies across the world that we were able to develop a general model which showed that about 55% of child deaths are due to the potentiating effects of malnutrition on infectious disease. The other statistic is that 86% of these infections begin in children who have only mild to moderate malnutrition. So I believe we should become, in public health terms, the consensus builders based on recognizing that we can use children’s weight for a general index of a community’s well-being and that we should now concentrate on reducing not only the number of severely malnourished but also target those with mild and moderate malnutrition. This agreement on public health could then be used to gain political agreement and action.

Rosenberg: This general approach is welcome. Homo sapiens lost the ability to synthesize vitamins early on in evolution so we have developed complex compensatory systems to minimize the damage from the relative deficiency of a single nutrient. Alternative and versatile pathways have developed to compensate so we can perhaps see only modest gains from manipulating individual nutrients.

Nutrition Reviews@, Vol. 58, No. 2 s73

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